Structural modifications of nile red carbon monoxide fluorescent probe: Sensing mechanism and applications

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Structural Modiﬁcations of Nile Red Carbon Monoxide Fluorescent

Probe: Sensing Mechanism and Applications

Dominik Madea, Marek Mart í nek, Lucie Muchova, Jir í Vana, Libor V í tek, and Petr Klan*

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ABSTRACT: Carbon monoxide (CO) is a cell-signaling molecule (gasotrans-

mitter) produced endogenously by oxidative catabolism of heme, and the

understanding of its spatial and temporal sensing at the cellular level is still an

open challenge. Synthesis, optical properties, and study of the sensing mechanism

of Nile red Pd-based CO chemosensors, structurally modiﬁed by core and bridge

substituents, in methanol and aqueous solutions are reported in this work. The

sensing ﬂuorescence “oﬀ−on” response of palladacycle-based sensors possessing

low-background ﬂuorescence arises from their reaction with CO to release the

corresponding highly ﬂuorescent Nile red derivatives in the ﬁnal step. Our

mechanistic study showed that electron-withdrawing and electron-donating core

substituents aﬀect the rate-determining step of the reaction. More importantly, the substituents were found to have a substantial

eﬀect on the Nile red sensor ﬂuorescence quantum yields, hereby deﬁning the sensing detection limit. The highest overall

ﬂuorescence and sensing rate enhancements were found for a 2-hydroxy palladacycle derivative, which was used in subsequent

biological studies on mouse hepatoma cells as it easily crosses the cell membrane and qualitatively traces the localization of CO

within the intracellular compartment with the linear quantitative response to increasing CO concentrations.

CO sensing can be based on its interaction with transition-

metal species, and two major detection mechanisms are

recognized. The ﬁrst process is a palladium-catalyzed

substitution (Tsuji−Trost) reaction,²⁶⁻³⁴originally used for

Pd⁰detection.^35,36It is a catalytic, thus very sensitive process,

but it requires introducing a second reaction component, a Pd^II

salt (Scheme 1a). The second mechanism is the incorporation

of CO into a palladacycle-based sensor that results in the

liberation of Pd as a heavy atom responsible for initial

ﬂuorescence quenching of the sensor (Scheme 1b).³⁷⁻⁴¹The

latter systems are less sensitive but do not require the presence

of any other reagent. The applications of palladacycle-based

CO sensors are usually limited by their lower sensitivity, low

aqueous solubility, and thermal instability. Only a few other

reported CO chemosensors use diﬀerent sensing mechanisms.

Some of them are based on a steric hindrance associated with

binding of CO to a transition metal.⁴²⁻⁴⁴Other examples of

CO detection involve an azidocarbonylation reaction in the

presence of a Pd catalyst^45,46or utilize CO to reduce a nitro

group into an amino group in the presence of a transition

metal as catalyst.⁴⁷⁻⁵¹

INTRODUCTION

■

Carbon monoxide (CO) is known for its toxic eﬀects in

mammals because its binding aﬃnity for hemoglobin is over

200 times greater than that of molecular oxygen.^1,2At low

concentrations, CO acts as a cell-signaling molecule (gaso-

transmitter),³and it is produced endogenously by the oxidative

catabolism of heme-by-heme oxygenase (HMOX-1) and, to

some extent, by lipid peroxidation as well as gut microbiota.⁴It

has been evidenced that CO is biologically relevant; it exhibits

antiapoptotic, anti-inﬂammatory, antiproliferative, and anti-

coagulation eﬀects⁵⁻⁷and prevention of tissue damage and

cardiovascular pathology,⁸although the mechanisms are still

not well understood. To utilize these potentially chemo-

preventive and/or therapeutic eﬀects, CO-releasing molecules

(CORMs) have been developed as artiﬁcial tools to deliver

therapeutic amounts of CO into the cell using various

initiation approaches.^7,9−16

The increasing interest in CO as a signaling molecule has led

to attempts to develop sensors, which detect, monitor, and

quantify this gasotransmitter in biological samples, cells, and

tissues. Many methods for the detection of exo- and

endogenous CO in cells or living organisms have already

been developed, especially in the past decade. Besides

electrochemical,^17,18infrared,¹⁹surface-enhanced Raman spec-

troscopy,²⁰and colorimetric²¹⁻²³methods, several ﬂuorescent

molecular chemosensors have been reported.^24,25Chemo-

sensors can provide a high spatio-temporal resolution of

analytes.

For this work, we selected one of the most promising

ﬂuorescent “oﬀ−on” probes based on a Nile red ﬂuorophore

Received: November 28, 2019

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subsequently, on the ﬂuorescent probe release triggered by its

reaction with CO using NMR, high resolution mass

spectrometry (HRMS), and electrospray ionization mass

spectrometry (ESI-MS) techniques. Finally, the biological

usability of the selected sensor is demonstrated.

Scheme 1. (a) Tsuji−Trost-Based Fluorescent CO Sensors;

(b) Palladacycle-Based Fluorescent CO Sensors (a

Simpliﬁcation)

RESULTS AND DISCUSSION

■

Synthesis. 1,4-Epoxynaphthalene derivative 1 was treated

with BF₃to give synthetic intermediate 7-ﬂuoronaphthalen-1-

ol (2), formed as a major product in 44% yield along with its 6-

ﬂuoro isomer 3 (Scheme 3) according to Repine and co-

workers.⁵²Another building block, 8-ﬂuoronaphthalen-1-ol 6,

was prepared via a three-step reaction reported by Cibulka⁵³

and Zhu,⁵⁴involving compounds 4 and 5, respectively, in the

overall chemical yield of 29%. Aminophenols (7−10),

synthesized as shown in Scheme 4, were used for the

preparation of nitrosophenols 11a−c using general procedure

A, described in the Experimental Section (Scheme 5). The

condensation of 11a−c with the corresponding napthalene-1-

ols in dimethylformamide (DMF) gave Nile red derivatives

12−14 (Scheme 6). Their isolated chemical yields were

moderate to low (5−30%); they were lower especially in the

case of more polar derivatives 13a,b and 14a,b after an

indispensable repetitive puriﬁcation process (column chroma-

tography and recrystallization). The 3-methoxy derivative 15

was prepared by a subsequent methylation of 12d in a good

yield (69%).

(9-diethylamino-5-benzo[a]phenoxazinone) reported by Lin

and co-workers,⁴⁰and we modiﬁed its structure to overcome

some of its drawbacks, such as its lower sensitivity and poor

solubility in water, to propose an improved CO sensor for

human biology applications (Scheme 2). Our strategy involved

Palladacycles 16−19 were synthesized from compounds

12−15 by C−H bond activation with Pd(OAc)₂in acetic acid

(Scheme 7) using modiﬁed procedures reported in the

literature.^40,55,56The amount of Pd(OAc)₂(1−2 equiv) used

was optimized. Residual Pd^IIsalt and/or unreacted starting

material was removed by recrystallization to give palladacycles

in 40−90% (16a−c, 19) and 30−60% (17a,b and 18a,b). The

synthesis of 4-ﬂuoro derivative 16e was not successful; only a

mixture of unidentiﬁed products was observed. The mecha-

nism of C−H bond activation⁵⁷resembles the electrophilic

aromatic substitution; deactivation of a π-system by electron-

withdrawing ﬂuorine in the para position to the C−Pd bond

may be detrimental for this reaction. 3-Fluoro derivative 16c

was successfully prepared in a lower yield (41%). Triﬂuor-

oacetate-bridged palladacycle 20 (Scheme 8) was prepared in a

similar manner to palladacycles 16−19 in a good yield (60%)

using triﬂuoroacetic acid as a solvent.

Scheme 2. Nile Red-Based CO Sensor (R₁, R₂= H, X =

CH₃COO)⁴⁰and Its Modiﬁcations Reported in This Work

modiﬁcations of the bridged ligand X, diﬀerent electron-

withdrawing or electron-donating groups in the 2−4 positions

of the Nile red core, and addition of water-solubilizing groups.

Because a sensing mechanism of Nile red chemosensors has

not been studied yet, we investigated the substituent eﬀects on

the mechanism of CO incorporation into a palladacycle and,

Palladacycles usually exist as dimers in both the solid state

and solutions.⁵⁸The NMR spectra of water-soluble derivatives

(17a,b, 18a,b) in D₂O were not resolved even at lower

temperatures. The HRMS spectra of 16−20 (Figures S67 −

Scheme 3. Synthesis of 7- and 8-Fluoronaphthalene-1-ols (2 and 6)

B

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Scheme 4. Synthesis of Aminophenols 8 and 10

Scheme 5. Synthesis of Nitrosophenol Hydrochlorides 11a-c

Scheme 7. Synthesis of Nile Red Palladacycles

S74) showed signals of both the monomeric and dimeric forms

in agreement with the literature,⁴¹although the dimer signals

were negligible or even absent in some cases (Figures S68 and

S72 −S74). To gain further evidence that the prepared

complexes exist as dimers, the bridge-splitting reaction of

complexes 16a−c and 20 with pyridine in dry dichloro-

methane as a noncoordinating solvent was studied.⁵⁹Upon

pyridine addition, the solutions of all palladacycles exhibited

changes in their absorption spectra. The corresponding

equilibrium constants were determined by titration and global

ﬁtting of the absorbance changes (Table 1 and Figures S80 −

S82). The equilibrium constant for the triﬂuoroacetate

complex 20 could not be calculated because the bridge

splitting with pyridine was quantitative.

Reaction of CO with Palladacycles 16−20. Initially, we

identiﬁed products of the reaction of CO with palladacycles

dissolved in methanol or phosphate-buﬀered saline (PBS). The

less polar probes (16a−c, 19, 20) are not suﬃciently soluble in

water; thus, their reactions were studied in methanol. A

solution of a palladacycle (16a−c) in methanol was stirred

under a CO atmosphere for 16 h to give a methyl ester of

carboxylic acid (21a−c) in quantitative yields (Scheme 9).

Scheme 8. Synthesis of Triﬂuoroacetate-Bridged

Palladacycle 20

These products, along with Pd⁰black and acetic/triﬂuoroacetic

acid byproducts, were formed via Pd-mediated carbon-

ylation.⁶⁰⁻⁶²The reactions of palladacycles 19 and 20 resulted

in a mixture of carbonylated (22 and 21a) and reduction (15

and 12a) side products, respectively, in the ratios of ∼1.5 : 1

(Figures S53 and S54). Therefore, compounds 19 and 20 were

excluded from further studies.

More polar probes (17a,b, 18a,b) are suﬃciently soluble in

PBS. A solution of a palladacycle (17a, ∼10 μM) in PBS was

Scheme 6. Synthesis of Nile Red Derivatives

C

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washed with PBS and visualized under a ﬂuorescence

microscope. Intensive ﬂuorescence was detected after treating

Hepa 1−6 cells only with 16b, whereas almost no signal and

only a distinct plasma membrane ﬂuorescence following

treatment with 17b and 18b were observed, respectively,

indicating a limited penetration of 17b and 18b into the

intracellular compartment (Figure S89). As a result, less polar

derivatives were selected for further experiments. It has been

reported that Pd^IIion inhibits enzyme activities at concen-

trations ranging from 20 to 500 mmol L⁻¹, attributed to the

binding of Pd^IIto enzymes, which leads to an altered peptide

conformation and destruction of catalytic activity.⁶³For

example, cellular function inhibition occurs at 100−400

mmol L⁻¹, whereas DNA synthesis is inhibited at 100−300

mmol L⁻¹.⁶⁴As the concentration of our sensor (10 μM) is

much lower than that in the studied systems, and most

importantly, any apparent toxicity of the sensor was not

observed in any our studies under our experimental conditions,

we assumed that toxicity of Pd^IIdoes not interfere with our

results.

Table 1. Equilibrium Constants for the Bridge Splitting of

16a−c and 20 with Pyridine in Dry Dichloromethane

c_M²

c_Dc_L²

K^a

=

compd

a

16a

16b

16c

20

3.8 0.1

nd

b

6.6 0.1

>10³

a

b

Determined at 20.0 0.1 °C. A pyridine complex is not stable.

Scheme 9. Reaction of CO with Nile Red Palladacycles in

Methanol and PBS

Optical Properties of Nile Red Derivatives and

Palladacycles. Nile red is a solvatochromic dye,⁶⁵and its

absorption and emission maxima and ﬂuorescence quantum

yields are considerably aﬀected by solvent polarity. Its

absorption and emission maxima exhibit strong bathochromic

shifts⁶⁶in polar solvents, whereas the ﬂuorescence quantum

yield decreases with an increased hydrogen-bonding power.⁶⁷

A low ﬂuorescence quantum yield in water is also attributed to

the H-type aggregation of the dye as a consequence of its low

solubility.⁶⁸

The absorption and emission maxima and ﬂuorescence

quantum yields of palladacycles 16a−c and 20 and Nile red

derivatives 12−15 and 21a (the products of CO reaction) are

summarized in Table 2 (see also Figures S59 −S64). The

stirred under CO atmosphere for 16 h, and the reaction

mixture was analyzed by high-performance liquid chromatog-

raphy (HPLC) and HRMS. The reduction product, a Nile red

derivative 13a, was formed in >80% yield (HPLC, HRMS;

Scheme 9 and Figure S58). This ﬁnding is in accordance with

the observed reactivity of an unsubstituted derivative 16a⁵⁶

and other palladacycles used as CO probes in aqueous

solutions.³⁸⁻⁴¹In contrast, a boron dipyrromethene (BODI-

PY)-derived Chang’s probe (Figure 1)³⁷gave a substituted

Table 2. Photophysical Properties of Nile Red Derivatives

and Palladacycles in Methanol

a

ε_max(10⁴dm³

λ_max(abs)

λ_max(em)

b

e

compd

mol⁻¹cm⁻¹

4.2

)

(nm)

Φ_F

12a

553

640

0.51, (0.38),⁷²

(0.40)⁶⁷

12b

12c

12e

13a

4.2

1.2

3.3

547

557

560

637

643

647

640, 659

0.50, (0.47)⁷³

0.44

0.46

c

0.44, 0.063,

d

3.0, 3.3

552, 564

0.091

c

0.54, 0.097,

d

13b

3.0, 2.6

549, 560

637, 655

Figure 1. BODIPY-Based Probe for CO as Reported by Chang and

Co-Workers.³⁷

0.10

c

d

14a

14b

15

16a

16b

16c

20

2.4, 2.6

2.0, 2.7

552, 571

546, 561

640, 654

637, 652

649

0.55, 0.19, 0.20

c

0.60, 0.13, 0.20

benzoic acid derivative instead. The reaction of CO with

nonpolar probes (16a−c, 19, 20) in PBS (using various

amounts of organic cosolvents, such as dimethylsulfoxide

(DMSO), tetrahydrofuran (THF), acetonitrile, 10−50%) was

inhibited probably because of a large amount of coordinating

organic cosolvent, and the formation of many side products

was observed. Still, reduction was the major reaction pathway

(Scheme 9).

Interaction of Sensors with Mouse Hepatoma Cells.

To select a few derivatives for spectroscopic and kinetic

studies, the interaction of cells with a CO sensor was evaluated

following the exposure of mouse hepatoma (Hepa 1−6) cells

to Nile red probes without (16b) and with water-solubilizing

sulfonic (17b, 18b) groups. After incubation, the cells were

4.4

552

610

0.49

0.080

h

3.8

715

h

f

h

fg

h

fg

3.4

3.3

610, 608 718, nd ^,0.010, nd ^,

h

630

616

561

717

716

635

0.065

0.080

0.54

h

2.5

21a

4.0

a

The values ε for palladacycles were calculated using the molar

b

masses of the dimers. λ_ex(palladacycles 16a−c, 20) = 600 nm, λ_ex

(Nile red derivatives) = 520 nm. In PBS (pH = 7.4, 10 mM, I = 0.1

M). In water. Φ_Fwas determined in solutions with A(λ_ex) < 0.1; the

values in parentheses were taken from the literature. In PBS (pH =

7.4, 10 mM, I = 0.1 M) with 5% DMSO. nd = no ﬂuorescence

detected. In a methanol/dichloromethane mixture (9:1).

c

d

e

f

g

h

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absorption and emission maxima of Nile red derivatives and

palladacycles in methanol were found not to be signiﬁcantly

inﬂuenced by the substituents. The ﬂuorescence quantum

yields of all Nile red derivatives in methanol were comparable

(0.45−0.55), whereas those of palladacycles were low due to

the quenching by a heavy atom (Pd).⁶⁹The ﬂuorescence

quantum yield of 2-hydroxy derivative 16b (0.01) is almost an

order of magnitude lower than that of the other derivatives,

which is very important when this system is considered as a

potential chemosensor (the background initial ﬂuorescence of

an oﬀ−on sensor should be as low as possible). Interestingly,

some reported Nile red Pd complexes exhibited high

ﬂuorescence quantum yields (∼0.5), which was attributed to

the low-energy ligand-centered excited states that are not

inﬂuenced by nonemissive metal-centered excited states.^70,71

The absorption and emission maxima of all compounds in PBS

were bathochromically shifted compared to those in methanol

(Table 2). The ﬂuorescence quantum yields of water-soluble

derivatives (13a,b, 14a,b) increased with the number of

solubilizing groups attached to the molecule, which can be

explained by the presence of charged sulfonic groups that make

the compounds less predisposed to aggregation.⁷²

Thermal Stability and Photostability. Sensors 16a−c

and 20 are not stable in methanol in the dark. They slowly

decompose to give the corresponding Nile red derivatives, that

is, the products of reduction (see the Experimental Section).

The rate of 16b decomposition at room temperature was

9.7 × 10⁻⁶s⁻¹; thus, the half-life of the sensor in this solvent

was ∼20 h (Figure S65). The thermal stability and photo-

stability of palladacycle 20 (c = 10 mM) were also examined in

PBS (5% DMSO) by emission spectroscopy. The compound

was found to be stable for 8 h in the dark (Figure S66);

however, it degrades photochemically under aerobic con-

ditions. Irradiation of the sample (20, 10 μM, 5% DMSO in

PBS) with 590 nm light-emitting diodes (LEDs) led to a 3-fold

ﬂuorescence enhancement over 4 h until the signal leveled oﬀ,

suggesting that singlet oxygen, which can be generated by

triplet−triplet annihilation of triplet-excited 20 with ground-

state oxygen, might be responsible for the conversion of the

palladacycle into a ﬂuorescent Nile red derivative. In contrast,

compound 20 was found to be suﬃciently photostable under

anaerobic conditions.

Reaction Kinetics of CO Sensing. The course of the

reaction of palladacycles with CO was ﬁrst studied by

absorption spectroscopy under pseudo-ﬁrst-order conditions

with an excess of CO (CO atmosphere) and a nucleophile

(methanol). The absorption spectra changes during the

reaction of derivatives 16a−c with CO are shown in Figure

2, where the spectra in red are always assigned to an initial

palladacycle. After a methanolic solution was purged with pure

CO, the formation of an intermediate (green line) was

observed within a few seconds. Thus, this ﬁrst step must be

very fast, whereas the subsequent transformation of these

intermediates to the ﬁnal methyl ester products (blue

spectrum) is considerably slower.

Figure 2. Reactions of palladacycles (c ∼ 10 μM) under the CO

atmosphere in methanol studied by absorption spectroscopy at

20.0 °C.

rate constant (ﬁrst order) was used to ﬁt the data (Table 3).

The kinetics of triﬂuoroacetate-bridged palladacycle 20 cannot

Table 3. Kinetic Data for the Rate-Determining Step in the

Reaction of Nile Red Palladacycles in Methanol under CO

a

Atmosphere

compd

k_AB(10⁻³s⁻¹

)

τ_AB(min)

16a

16b

16c

7.3 0.2

13.0 0.5

1.24 0.05

2.3 0.1

1.3 0.1

13.4 0.5

a

The rate constants were obtained by target analysis using

spectroscopic data from the reactions of palladacycles (c ∼ 10 μM)

with CO (1 atm) in methanol at 20.0 0.1 °C. An A → B kinetic

model was used in all cases.

be directly compared to the other derivatives because the

simultaneous formation of methyl ester 21a and a reduction

product 12a was observed. A branching model was used for

ﬁtting (see Figures S87 and S88).

To unravel the structure of a long-lived intermediate, we

performed the same experiment with palladacycle 16a, but the

solution was purged with argon after CO addition (Figure 3).

A slow disappearance of the absorption band of an

intermediate at 596 nm and the appearance of a band that

The recorded kinetic data related to the transformation of an

intermediate to the product were subjected to target analysis.

Singular value decomposition (SVD)⁷⁴of the kinetic data was

performed to distinguish the number of individual species

involved. We concluded from the magnitude of singular values

(Figure S83) and shapes of the spectral and trace vectors that

there are two contributing components in the case of

derivatives 16a−c. A simple A → B kinetic model with one

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concentrations used in spectroscopic experiments but not at 2

orders of magnitude higher concentrations in an NMR tube.

In addition, the reaction kinetics of 16a−c and 20 (c ∼1

μM) in methanol under a CO atmosphere was studied by

emission spectroscopy. The ﬂuorescence enhancement due to

the formation of ﬂuorescent products are usually calculated

from the time-dependent emission spectra (Figure 4a) as the

Figure 3. Reversible binding of CO to 16a in methanol. A solution of

16a (thick red line) was purged with CO for 15 s (thick green line); it

was allowed to react for 30 s (black line) and then purged with argon

(gray lines) for 4 min (thick blue line).

corresponds to the initial complex (at 613 nm) were observed

upon excessive argon purging, suggesting that CO binding

occurs in the ﬁrst step and is reversible. Because the reaction

was conducted in methanol, we also detected the formation of

methyl ester 21a during purging the solution with argon. The

ﬁnal spectrum (after 4 min of argon purging) can be expressed

as a linear combination of the spectra of starting material 16a

and product 21a (Figure S78).

A reversible binding of CO in a dichloromethane solution

was also observed for triﬂuoroacetate-bridged complex 20

(Figure S76) but not for acetate-bridged palladacycles 16a−c.

This ﬁnding is in accordance with the bridge-splitting

equilibrium constants found for pyridine (Table 1), where

the derivative 20 reacted with pyridine quantitatively, but a

large excess was necessary in the case of acetate derivatives

16a−c (triﬂuoroacetate is a better leaving group than acetate).

Ligand exchange of CO by pyridine in complex 20 was

monitored by UV−vis spectroscopy, and the species involved

were directly detected by ESI-MS. In addition, the collision-

induced dissociation (CID) of a mixed pyridine/CO-

coordinated complex (12a-H)₂Pd₂(pyridine)(CF₃COO⁻)-

(CO) led to the loss of CO (Figure S77).

To get a deeper insight into the reaction mechanism, we

attempted to identify the reaction intermediates observed in

spectroscopic experiments (Figure 2) by ESI-MS.⁷⁵No species

were detected under these conditions, most probably because

of their poor stability even under soft ionization conditions.⁷⁶

However, the addition of pyridine led to the formation of

detectable complexes. For example, the reaction of complex

16a gave a mixed CO/pyridine adduct along with the

formation of the ﬁnal carbonylation product 21a (Figure

S79). Therefore, we assign the intermediates (green spectrum,

Figure 2) to the CO-coordinated complex according to the

following considerations: (a) The CO binding is reversible in

both methanol (Figure 3) and dichloromethane (Figure S76);

(b) CO/pyridine adducts were detected by ESI-MS, and they

release CO under the CID experiments (Figures S77 and S79);

and (c) no other reaction intermediates were detected.

This reaction mechanism was also studied by NMR. The

addition of dichloromethane-d₂as an inert cosolvent was

necessary for dissolving larger amounts of the starting material,

and it had no eﬀect on the product formation. Unfortunately,

only the decay of palladacycle 16a to give product 21a was

observed (Figure S57). The explanation is trivial; the solubility

of CO in methanol (∼10⁻²M)⁷⁷is suﬃcient to convert

starting palladacycle to a CO-coordinated intermediate at low

Figure 4. (a) Representative time-dependent emission spectra of the

reaction of 16a (c ∼ 1 μM, λ_ex= 520 nm; measured for ∼2 h) under

CO atmosphere in methanol. (b) Time evolution of I for 16a−c.

̅

F

ratio of ﬂuorescence intensities at the corresponding wave-

lengths (typically emission maxima).³⁸⁻⁴¹In this work,

complete spectra were used in the calculation to improve the

signal-to-noise ratio (eq 1) and to clearly distinguish the

signals of the initial and end states. The ﬂuorescence

enhancement (I ) is proportional to the ratio of the

̅

F

ﬂuorescence quantum yields of the starting palladacycle and

the product; thus, the lower the ﬂuorescence quantum yield of

palladacycle, the higher the ﬂuorescence enhancement.

λ

λ₁

²I_F(λ, t)dλ

∫

Φ_F,p× ε_p(λ_ex)

I_F=

∝

λ

λ₁

²I_F(λ, 0)dλ

Φ_F,c× ε (λ_ex)

c

∫

(1)

where I_F(λ,t) is the ﬂuorescence intensity at time t and

wavelength λ; Φ_F,pand Φ_F,care the ﬂuorescence quantum

yields of the product and the corresponding complex,

respectively; and ε_p(λ_ex) and ε_c(λ_ex) are the molar absorption

coeﬃcients at the excitation wavelength of the related species.

Our results show that 16b exhibits a much greater

ﬂuorescence enhancement than I of the other derivatives

̅

F

(Figure 4b), which is simply related to a very low ﬂuorescence

quantum yield of the starting compound 16b (Φ_F= 0.01 in

methanol; Table 2), being responsible for a lower signal-to-

noise ratio, whereas Φ_Fof 21a is comparable to Φ_F’s of other

Nile red derivatives listed in Table 2. The rate of CO sensing

followed the trend of 16b > 16a > 16c (Table 3). The

electron-donating hydroxyl group (16b) increased and the

electron-withdrawing ﬂuoride (16c) decreased the reaction

rate by factors of approximately 1.8 and 6, respectively,

compared to that of an unsubstituted derivative 16a (Table 3).

F

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Scheme 10. Proposed Mechanism of CO Sensing in Methanol

Reaction Mechanism. Our experiments provided evi-

dence that the CO coordination is a reversible but rapid

reaction step to give a long-lived carbonyl complex as an

intermediate. No other reaction intermediate was detected or

indicated in our kinetic spectroscopic experiments. Therefore,

the subsequent process must be rate-determining, followed by

rapid reaction steps to give the ﬁnal products. If a CO insertion

into palladacycles is the rate-determining step (Scheme 10),

the reaction should be sensitive to the nature of the migrating

group. It has been shown that electron-donating groups

accelerate CO insertion into palladacycles and vice versa.⁷⁸We

observed the same trend, and we propose that the CO

insertion is the rate-determining step (Table 3). Therefore, the

sensing mechanism most likely involves CO coordination, CO

insertion, and the subsequent reductive elimination steps

(Scheme 10).

Figure 5. Fluorescence intensity increase of 16b (λ_ex= 530 nm, λ_em

=

645 nm) in the presence of CO released from a photoCORM at

concentrations of 62.5, 125, 500, and 1000 μM after 5 min irradiation

with an LED source (400 nm) in Hepa 1−6 cells on a microplate

reader. The values represent mean SD (n = 8).

The overall ﬂuorescence enhancement I is the major feature

̅

F

for sensing applications where the detection sensitivity is a key

issue. Incomparably, the greatest I was found for the

̅

remained constant to clearly demonstrate the relationship

between ﬂuorescence intensity and CO concentration.

F

derivative 16b (Figure 3), considerably exceeding the

sensitivity of a nonsubstituted derivative 16a reported earlier.⁴⁰

As this sensing process is also the fastest, 16b represented the

best candidate for the subsequent biological studies.

CONCLUSIONS

■

Diﬀerently substituted Nile red palladacycles with core and

bridging ligand modiﬁcations were synthesized as CO

ﬂuorescent oﬀ−on sensors in aqueous solutions. We

determined their optical properties and reactivities with CO

using time-dependent absorption and ﬂuorescence spectros-

copies. The longest-living intermediate in CO sensing was

found to be a CO-coordinated complex, followed by CO

insertion as the rate-determining step. An electron-donating

core substituent in 16b was found to increase the overall

reaction rate and the overall ﬂuorescence enhancement. The

sensing reaction of this compound was then successfully tested

in biological samples using ﬂuorescence microscopy, which

enabled in situ visualization and localization of metabolic

processes employing CO within the speciﬁc cellular and

subcellular compartments. From this perspective, the sensor

16b represents a very promising molecule easily crossing the

plasma membrane and qualitatively tracing the localization of

CO within the cell interior with the linear quantitative

response to increasing CO concentrations. Further in vitro

and in vivo studies are needed to uncover the whole biological

potential of this probe.

Biological Experiments. A biological usability of CO

sensing was investigated with sensor 16b (10 μM) in the cell

culture of mouse hepatoma (Hepa 1−6) cells after incubation

in the presence of a photoactivatable CO-releasing molecule

(photoCORM; 4,8-dimethoxy-6,6-dimethyldibenzo[b,f ]-

cyclopropa[d]silepin-1(6H)-one), which activates CO produc-

tion upon irradiation.⁷⁹The cells were washed with PBS after

reaching an 80% conﬂuence and were incubated with

increasing concentrations of photoCORM. After activation of

CO release upon irradiation at 400 nm, followed by 15 min

incubation in the dark, the ﬂuorescence intensity was

determined using a microplate reader, and the samples were

visualized by ﬂuorescence microscopy. A linear increase in the

ﬂuorescence was observed in the photoCORM concentration

range of 60−1000 μM (Figure 5). These data were in

concordance with live-cell CO imaging using ﬂuorescence

microscopy, where a substantial CO-dependent increase in the

ﬂuorescence intensity and intracellular accumulation of 16b

were observed (Figure 6). In this set of experiments, the probe

concentration as well as the exposure time during imaging

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IR spectra were recorded on a Fourier transform spectrometer using

solid samples. HRMS spectra were obtained on a triple-quadrupole

ESI mass spectrometer in a positive and/or negative mode. UV−vis

spectra were measured in 10.0 mm quartz ﬂuorescence cuvettes using

a UV−vis spectrometer. Emission spectra were recorded on a

luminescence spectrometer in 10.0 mm quartz ﬂuorescence cuvettes.

HPLC (C-18 column) analysis was performed by gradient elution

(50:50 to 0:100 of solutions A:B, where A = 0.1 M NH₄OAc in H₂O

and B = 0.1 M NH₄OAc in methanol). All column chromatography

procedures were performed on columns packed with silica gel (63−

200 μm). Thin-layer chromatography (TLC) was performed using

silica gel plates (0.2 mm thickness) and visualized under a UV lamp

(254 nm, 366 nm). All solvents and chemicals were used as purchased

or puriﬁed/dried by standard procedures when necessary. Unless

otherwise speciﬁed, all procedures were carried out under a nitrogen

atmosphere. Reaction mixtures were heated using a hot plate with

magnetic stirring in an oil bath.

Determination of Fluorescence Quantum Yields. Fluores-

cence quantum yields were determined as the absolute values using an

integration sphere. The quantum yields were measured three times

and then averaged for each sample. The concentrations of the

solutions were kept low (A < 0.1). The ﬂuorescence quantum yields

were determined in methanol, PBS (pH = 7.4, 10 mM, I = 0.1 M), or

water. The 520 nm excitation wavelength was used for the

determination of the ﬂuorescence quantum yields of Nile red

derivatives (12a−c, 12e, 13−15), and the 600 nm excitation

wavelength was used for Nile red-derived palladacycles (16a−c,

20). The wavelength of 600 nm was chosen to eliminate the eﬀect of

ubiquitous impurities of Nile red derivatives, whose molar absorption

coeﬃcient at 600 nm is suﬃciently small.

Determination of Bridge-Splitting Equilibrium Constant.

Two solutions of the same concentrations (∼10 μM) of palladacycles

16a−c or 20 in dry dichloromethane were prepared, and the

corresponding amount of pyridine was added to one of them. The

solution of a pure palladacycle was then titrated with the other

solution, and absorption spectra were recorded upon each addition.

The pyridine concentration-dependent spectra were then subjected to

global ﬁtting using the corresponding equilibrium model (Supporting

Information, page S79; Figures S80 −S82).

Reaction Kinetics of Nile Red Palladacycles (16a−c, 20) with

CO in Methanol. All solutions were freshly prepared before the

measurements. The palladacycles decompose over time in both

DMSO and methanol (see below).

Absorption Spectroscopy. A solution (∼10 μM) of Nile red

palladacycle (16a−c, 20) in methanol was prepared by dissolving the

pure compound in a drop of dry dichloromethane (∼0.1 mL) and

dilution with dry methanol. This solution was then ﬁltered through a

syringe ﬁlter prior to each measurement to remove any undissolved

solids. A ﬁltered solution (2 mL) was added to a cuvette (total

volume, 4.6 mL) equipped with a stir bar and silicon septum. The

cuvette was inserted into a spectrometer, and the solution was stirred.

The absorption spectrum before the addition of CO was recorded,

and subsequently, the solution was purged with pure CO for 15 s

while the air was vented out via another syringe. Both syringes were

removed and time-dependent absorption spectra were recorded in a

thermostated cuvette holder setup at 20.0 0.1 °C for ∼ 2 h under

stirring.

Emission Spectroscopy. A stock solution of palladacycle (16a−

c) was prepared in the same manner as that for the absorption kinetic

measurements. An ∼1 μM solution (A < 0.1) was prepared from the

stock solution by dilution, and one part (2 mL) was added to a

cuvette (total volume, 4.6 mL) equipped with a stir bar and a silicon

septum. The cuvette was placed into a ﬂuorimeter and equipped with

a magnetic stirrer. CO was introduced to a solution in the same

manner as was described in the case of absorption spectra

measurements. Time-dependent emission spectra (560−800 nm)

were recorded every 30 s (dwell time, 0.08 s; 2 nm step; one repeat)

for ∼2 h with the excitation wavelength of 520 nm at room

temperature.

Figure 6. Fluorescence microscopy images of 16b with increasing

concentrations of CO (62.5, 500, and 1000 μM (a−c); see the

footnotes in Figure 5) in Hepa 1−6 cells at ﬁxed exposure times. The

scale bars represent 20 μm.

EXPERIMENTAL SECTION

■

Materials and Methods. Melting points were measured on a

noncalibrated Koﬂer’s hot-stage melting-point apparatus. NMR

spectra were obtained on 75 and 126 MHz (¹³C), and 300 and 500

MHz (¹H) spectrometers in chloroform-d, dimethylsulfoxide-d₆,

methanol-d₄, water-d₂, and dichloromethane-d₂, and they were

referenced to the residual peak of the (major) solvent except for

those of ¹⁹F NMR. All NMR measurements were conducted at 30 °C.

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Thermal Stabilities in Methanol and Rates of Decom-

position. A solution of 16a (∼5 mg) in methanol (20 mL) was

reﬂuxed under an air atmosphere for 16 h. Palladium black was

formed in the course of the reaction as a black precipitate. The

suspension was ﬁltered through a pad of Celite and evaporated. The

crude residue was analyzed by ¹H NMR. The formation of the parent

After cooling to room temperature, the mixture was ﬁltered through a

pad of Celite, and the ﬁltrate was concentrated on a rotary evaporator

to a volume of ∼30 mL. The mixture was quenched with water (30

mL) and extracted with diethyl ether (3 × 50 mL); brine (30 mL)

was added to facilitate separation of the layers. The organic extracts

were combined, dried over anhydrous MgSO₄, and evaporated under

reduced pressure to aﬀord the title compound as an amber oil, which

was subsequently used in the next step without further puriﬁcation.

Yield 7.00 g (83%). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.14 (dd,

J = 7.8, 4.6 Hz, 1H), 7.08−6.96 (m, 3H), 6.63 (ddd, J = 9.8, 7.8, 2.3

Hz, 1H), 5.69 (s, 2H). ¹⁹F NMR (282 MHz, CDCl₃): δ (ppm)

−117.66 (ddd, J = 9.7, 7.7, 4.6 Hz, 1F). The spectroscopic data are

consistent with those reported in the literature.⁸³

1

Nile red (12a) along with acetic acid was evident by H NMR.

The rate of thermal decomposition of 16b in methanol was studied

by time-dependent ﬂuorescence spectroscopy. The solution of 16b in

methanol (c ∼ 20 μM) was prepared and transferred to a ﬂuorescent

cuvette equipped with a septum. Time-dependent ﬂuorescence

spectra (dwell time, 0.2 s; 1 nm step, two repeats) were measured

every ∼10 min over the course of 16 h at room temperature with an

excitation wavelength of 520 nm (Figure S65a). The decomposition

rate constant was determined from the concentration change of the

product 12b (at 630 nm; Figure S65b).

Thermal Stability and Photostability in PBS. A solution of

compound 20 (10 μM) in PBS (5% DMSO, 10 mM, I = 0.1 M) in a

capped cuvette equipped with a silicon septum was irradiated by 590

nm LEDs in a cuvette holder for 8 h. The emission spectra were taken

every 1 h (Figure S66). For deoxygenated conditions, the same

experiment was performed, but the solution of the complex was

purged with argon for 1 min before the irradiation started (Figure

S66).

7-Fluoronaphthalen-1-ol (2). The compound was prepared

according to a known procedure.⁵²Diethyl ether·BF₃(5.6 mL, 45.3

mmol) was added dropwise to a solution of crude 6-ﬂuoro-1,4-

dihydro-1,4-epoxynaphthalene (1, 7.00 g, 43.2 mmol) in dry

dichloromethane (90 mL) at 0 °C. After this addition (30 min),

the mixture was allowed to warm to room temperature and then

stirred for additional 2 h. The resulting solution (dark brown) was

washed with water (3 × 80 mL). The organic layer was separated,

dried with anhydrous Na₂SO₄, and the solvent was evaporated under

reduced pressure to yield a dark orange crystalline mass. The mixture

was chromatographed ﬁrst with dichloromethane/hexane (1:1, v/v)

and then with dichloromethane as a mobile phase to yield the mixture

of 7- and 6-ﬂuoro (3) isomers, in the ratio of ∼14:1 (from integration

of signals in ¹⁹F NMR spectrum). The solid was then recrystallized

from boiling chloroform to produce crystals, which were ﬁltered and

washed with hexane (10 mL) to yield a white cotton candy-looking

NMR Kinetics. A solution of 16a (3.4 mg) in CD₃OD/CD₂Cl₂

(0.6 mL, 4:2, v/v) was prepared in an NMR tube. The air above the

solution was exchanged by a pure CO atmosphere, and the solution

1

was shaken. Time-dependent H NMR spectra were then acquired

(Figure S57).

1

Biological Experiments. Hepa 1−6 mouse hepatoma cell line

(ATCC CRL-1830) was grown in 48-well plates according to the

manufacturer′s instructions. The cells were kept at 37 °C and 5% CO₂

atmosphere throughout the experiment. Fluorescence was measured

using a multidetection microplate reader at λ_ex= 530 nm and λ_em=

645 nm, and the cells were visualized under a ﬂuorescence microscope

(emission ﬁlter: 420 nm). The pictures were taken under a constant

exposure time of 100 ms.

crystals with a characteristic naphthol aroma. Yield 3.10 g (44%). H

NMR (300 MHz, CDCl₃): δ (ppm) 7.86−7.75 (m, 2H), 7.44 (d, J =

8.3 Hz, 1H), 7.34−7.21 (m, 2H), 6.83 (d, J = 7.5 Hz, 1H), 5.16 (s,

1H). ¹⁹F NMR (282 MHz, CDCl₃): δ (ppm) −114.66 (ddd, J = 10.3,

8.5, 5.6 Hz, 1F). ¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 160.6

(d, ¹J_C−F= 245.1 Hz), 151.1 (d, J_C−F= 5.4 Hz), 132.0, 130.2 (d, J_C−F

= 8.8 Hz), 125.3 (d, J_C−F= 9.0 Hz), 125.1 (d, J_C−F= 2.5 Hz), 120.8

(d, J_C−F= 1.1 Hz), 117.0 (d, J_C−F= 25.4 Hz), 109.5, 105.8 (d, J_C−F

=

Global Fitting. Diﬀerent data (either time-resolved spectroscopic

data from the kinetic measurements or concentration-dependent

spectroscopic data from the determination of equilibrium constants),

represented by a matrix D = CS^T+ E, were decomposed into the

concentration C and spectra S^Tmatrices with an error E by a variable

projection approach.⁸⁰The corresponding kinetic or equilibrium

model with nonlinear parameters Θ was applied as a model for the

calculation of the concentration matrix C C(Θ). The spectra matrix

S^Tis conditionally linear (S^T C⁺D) on C, where C⁺ (C^TC)⁻¹C^T

is the Moore−Penrose pseudoinverse. Minimization of the sum of

squares of residuals ∥CS^T− D∥₂²= ∥(CC⁺− I)D∥²₂for the

parameters deﬁned in a corresponding model by the Levenberg−

Marquardt algorithm provided the best ﬁt of C and S^Twith optimized

parameters. Routines for the ﬁtting were programmed with the use of

an LMFIT package.⁸¹

ESI-MS Experiments. The experiments were performed with an

ion-trap mass spectrometer equipped with an HESI source operating

in the positive mode.⁸²The palladacycles were diluted by a solvent

(methanol or dichloromethane containing traces of pyridine) to the

corresponding concentration of 5 × 10⁻⁵M and were introduced to

an HESI source at a rate of 3 μL min⁻¹. The operating conditions

were set as follows: spray voltage, 3.7 kV; capillary voltage, 3 V; tube

lens oﬀset, 119 V; and heated capillary temperature, 150 °C. All of the

mass spectra were recorded from m/z 50 to 2000.

22.3 Hz). The spectroscopic data are consistent with those reported

in the literature.⁵²

1H-Naphtho[1,8-de][1,2,3]triazine (4). The compound was

prepared according to a known procedure.⁵³A solution of NaNO₂

(1.05 equiv, 4.72 g, 68.4 mmol) in water (27 mL) was added to a

solution of naphthalene-1,8-diamine (10.3 g, 65.1 mmol) in a mixture

of acetic acid (120 mL) and water (90 mL) at −6 °C under vigorous

stirring. The reaction temperature was raised to 10 °C; then, the

reaction mixture was diluted with water (30 mL) and stirred at 0 °C

for another 45 min. The brown precipitate was ﬁltered oﬀ, washed

with water (30 mL), and dried at room temperature to give a ﬁne red-

brown powder, which was a suﬃciently pure title compound for the

1

next step. Yield 10.0 g (91%). H NMR (300 MHz, DMSO-d₆): δ

(ppm) 13.15 (bs, 1H), 7.31−7.05 (m, 4H), 6.52 (d, J = 5.7 Hz, 2H).

¹³C{¹H} NMR (75 MHz, DSMO-d₆): δ (ppm) 136.0, 133.5, 129.1,

120.8, 118.4, 106.2 (bs). HRMS (APCI⁻) m/z: [M − H]⁻calcd for

C₁₀H₆N₃168.0567; found 168.0566. The spectroscopic data are

consistent with those reported in the literature.⁵³

8-Fluoronaphthalen-1-amine (5). The compound was prepared

according to a known procedure.⁵⁴1H-Naphtho[1,8-de][1,2,3]-

triazine (4, 3.38 g, 20.0 mmol) was added slowly in portions to a

solution of hydrogen ﬂuoride/pyridine (70% HF in pyridine, 33

equiv, 17 mL, 654 mmol) in a HD-PE ﬂask with magnetic stirring

while cooling in an ice bath. The reaction ﬂask was capped with a

septum, and a balloon with nitrogen was attached. The reaction

solution was stirred at room temperature for 7 days. The diluted

reaction mixture with water (100 mL) and ice (50 g) was neutralized

with an ice-cold solution of KOH (44.8 g, 799 mmol) in water (200

mL) while cooling in an ice bath. The solution was then extracted

with ethyl acetate (4 × 70 mL). The extracts were combined and

washed with brine (50 mL), dried using anhydrous Na₂SO₄, and

evaporated under reduced pressure to yield crude naphthylamine. It

was puriﬁed by column chromatography with dichloromethane as a

Synthesis. 6-Fluoro-1,4-dihydro-1,4-epoxynaphthalene (1). The

compound was prepared according to a known procedure.⁵²

A

suspension of furan (2.0 equiv, 7.5 mL, 104 mmol) and Mg turnings

(1.64 g, 67.4 mmol) in dry THF (80 mL) was prepared and heated to

60 °C. The Mg turnings were activated by the addition of iodine (∼20

mg) to the mixture. A solution of 1-bromo-2,4-diﬂourobenzene (10.0

g, 51.8 mmol) in dry THF (25 mL) was added dropwise to the

prepared mixture at a rate to maintain the suspension at a gentle reﬂux

(∼30 min). The reaction mixture was then stirred at 65 °C for 15 h.

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mobile phase to give a pink solid. Yield 1.60 g (50%). ¹H NMR (300

MHz, CDCl₃): δ (ppm) 7.52 (dd, J = 8.3, 0.9 Hz, 1H), 7.38−7.17 (m,

3H), 6.99 (ddd, J = 14.8, 7.6, 1.1 Hz, 1H), 6.67 (dd, J = 7.3, 1.2 Hz,

1H), 4.79 (s, 2H). ¹⁹F NMR (282 MHz, CDCl₃): δ (ppm) −116.06

(ddd, J = 14.7, 5.3, 2.4 Hz, 1H). ¹³C{¹H} NMR (75 MHz, CDCl₃): δ

39.6 mmol) in n-butanol (70 mL). The mixture was reﬂuxed for 4 h.

During the reﬂux, a gray precipitate was formed. The suspension was

cooled down to room temperature and ﬁltered, and solids were

washed with cold methanol (2 × 40 mL) and vacuum-dried. Yield

11.5 g (75%; purity 60% determined by integration of ¹H NMR

signals). Gray powder. The crude material was a mixture of the title

compound and a disubstituted derivative 10. This material was used

1

(ppm) 160.5 (d, J_C−F= 247.9 Hz), 142.6 (d, J_C−F= 3.4 Hz), 137.4

(d, J_C−F= 4.5 Hz), 127.8 (d, J_C−F= 1.6 Hz), 125.6 (d, J_C−F= 10.0

Hz), 124.6 (d, J_C−F= 3.9 Hz), 117.3 (d, J_C−F= 3.2 Hz), 113.6 (d, J_C−F

= 10.0 Hz), 110.2 (d, J_C−F= 2.0 Hz), 109.3 (d, J_C−F= 23.9 Hz).

HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₁₀H₉FN 162.0714; found

162.0714. The spectral data are consistent with those reported in the

literature.⁵⁴

1

directly in the next step without further puriﬁcation. H NMR (300

MHz, D₂O): δ (ppm) 7.49 (t, J = 8.2 Hz, 1H), 7.13−6.96 (m, 3H),

3.63 (t, J = 7.5 Hz, 2H), 3.07 (t, J = 7.3 Hz, 2H), 2.23 (p, J = 7.5 Hz,

2H). The spectroscopic data are consistent with those reported in the

literature.⁸⁷

8-Fluoronaphthalen-1-ol (6). A solution of NaNO₂(0.276 g, 4.00

mmol) in water (5 mL) was added dropwise to an ice-cooled

suspension of 8-ﬂuoronaphthalen-1-amine (5, 0.580 g, 3.60 mmol) in

a solution of concn H₂SO₄(98.0%, 4.6 mL, 84.9 mmol) in water (30

mL) under nitrogen atmosphere. The reaction mixture was stirred for

20 min at room temperature and then steam-distilled (excess of water

was added to the reaction mixture, 100 mL) under a nitrogen

atmosphere until no product appeared. The product was washed from

a condenser with ethyl acetate, and the distillate was extracted with

ethyl acetate (2 × 30 mL). Washings and extracts were combined,

washed with brine (30 mL), dried under anhydrous MgSO₄, and

3,3′-((3-Hydroxyphenyl)azanediyl)bis(propane-1-sulfonic acid)

(10). The compound was prepared by modiﬁcation of a known

procedure.⁸⁷The solution of crude 10 (60.0%, 1.0 equiv, 5.00 g, 13.0

mmol) and 1,3-propanesultone (2.5 equiv, 3.88 g, 31.8 mmol) in

DMF (40 mL) was heated to 145 °C for 1 h and then to 100 °C

overnight under stirring. The reaction was monitored by TLC

(methanol/ethyl acetate, 1:2, v/v). DMF was evaporated under

reduced pressure, and a residue was puriﬁed by silica get column

chromatography with the use of a gradient of acetone/H₂O (15:1 to

1:1, v/v) as a mobile phase to provide a dark brown solid of the title

compound. Yield 2.88 g (63%). ¹H NMR (300 MHz, D₂O): δ (ppm)

7.57 (t, J = 8.2 Hz, 1H), 7.23−7.01 (m, 3H), 3.83 (t, J = 8.0 Hz, 4H),

2.96 (t, J = 7.3 Hz, 4H), 2.01 (p, J = 7.5 Hz, 4H). The spectroscopic

data are consistent with those reported in the literature.⁸⁷

General Procedure A: Preparation of Nitrosophenols 11a−

c. Aminophenol (3-diethylaminophenol, 8 or 10, 90 mmol) was

dissolved in a mixture of water (20 mL) and concn HCl (50 mL). The

solution was cooled to 0 °C, and a solution of sodium nitrite (1.0

equiv, 90 mmol) in water (15 mL) was added dropwise over a period

of 30 min. The mixture of concn HCl and water (2:1, v/v) was added

when stirring was not possible (∼20 mL). Stirring was continued at 0

°C for 1 h. The suspension was ﬁltered, and the residue was dried

under vacuum to yield a brown to orange powder, which was

suﬃciently pure for the next step. In the case of water-soluble

derivatives (8 and 10), no precipitate was formed after the addition of

sodium nitrite; therefore, the solution was evaporated to dryness and

the residue was directly used in the next step.

1

evaporated to aﬀord pale yellow crystals. Yield 0.365 g (63%). H

NMR (300 MHz, CDCl₃): δ (ppm) 7.62 (dd, J = 8.3, 1.0 Hz, 1H),

7.45−7.39 (m, 2H), 7.35 (td, J = 8.0, 5.7 Hz, 1H), 7.09 (ddd, J = 15.1,

7.7, 1.0 Hz, 1H), 7.03−6.95 (m, 1.5H), 6.91 (s, 0.5H). ¹⁹F NMR (282

MHz, CDCl₃): δ (ppm) −121.39 (ddd, J = 26.5, 15.0, 5.3 Hz, 1H).

1

¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 159.5 (d, J_C−F= 242.3

Hz), 151.6 (d, J_C−F= 1.1 Hz), 137.2 (d, J_C−F= 3.5 Hz), 128.1 (d, J_C−F

= 1.5 Hz), 125.7 (d, J_C−F= 10.1 Hz), 124.9 (d, J_C−F= 3.7 Hz), 119.8

(d, J_C−F= 3.2 Hz), 113.9 (d, J_C−F= 7.3 Hz), 111.6 (d, J_C−F= 2.6 Hz),

109.7 (d, J_C−F= 22.8 Hz). HRMS (APCI⁻) m/z: [M − H]⁻calcd for

C₁₀H₆FO 161.0408; found 161.0407. The spectroscopic data are

consistent with those reported in the literature.⁸⁴

3-(Ethylamino)phenol (7). The compound was prepared by

modiﬁcation of a known procedure.⁸⁵Ethyl iodide (6.5 mL, 80.9

mmol) was added to a mixture of 3-aminophenol (2.0 equiv, 17.6 g,

162 mmol) and K₂CO₃(11.2 g, 80.9 mmol) in DMF (45 mL) under

a nitrogen atmosphere. The reaction mixture was heated to 100 °C for

2 h under stirring. The reaction mixture was poured into water (500

mL) and extracted with ethyl acetate (2 × 200 mL). The ethyl acetate

layer was washed with water (500 mL) and brine (300 mL). The

organic layer was dried with anhydrous Na₂SO₄and evaporated to

yield amber oil. The crude material was puriﬁed by column

chromatography with the use of a gradient of dichloromethane/

ethyl acetate (6:1 to 3:2, v/v) as a mobile phase. Yield 6.8 g (61%).

Pale yellow oil. ¹H NMR (300 MHz, CDCl₃): δ (ppm) 7.01 (t, J = 8.0

Hz, 1H), 6.26−6.12 (m, 2H), 6.11 (t, J = 2.3 Hz, 1H), 4.14 (bs, 2H,

NH + OH), 3.13 (q, J = 7.2 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H). The

spectroscopic data are consistent with those reported in the

literature.⁸⁶

5-(Diethylamino)-2-nitrosophenol Hydrochloride (11a). The

compound was prepared according to general procedure A from 3-

diethylaminophenol (14.9 g, 90.0 mmol). The pure sample was

prepared by recrystallization. The crude material was dissolved in a

small amount of methanol, ﬁltered, and the ﬁltrate was precipitated by

the addition of diethyl ether while cooling to −20 °C. The precipitate

was ﬁltered, washed with diethyl ether, and vacuum-dried. Yield 15.4

1

g (74%). Dark orange powder. H NMR (300 MHz, CD₃OD): δ

(ppm) 7.72 (d, J = 10.4 Hz, 1H), 7.21 (dd, J = 10.5, 2.3 Hz, 1H), 6.42

(d, J = 2.5 Hz, 1H), 3.93 (dq, J = 25.5, 7.2 Hz, 4H), 1.40 (t, J = 7.2

Hz, 6H). ¹³C{¹H} NMR (75 MHz, CD₃OD): δ (ppm) 167.2, 164.1,

145.9, 124.8, 121.0, 98.9, 14.6, 13.1. The spectroscopic data are

consistent with those reported in the literature.⁸⁸

3-(Ethyl(3-hydroxyphenyl)amino)propane-1-sulfonic Acid (8).

The compound was prepared according to a known procedure.⁷²3-

Ethylaminophenol (7, 6.35 g, 46.3 mmol) and 1,3-propanesultone

(1.1 equiv, 6.19 g, 50.7 mmol) were dissolved in i-propanol (50 mL)

and reﬂuxed under a nitrogen atmosphere for 3 h. During the reﬂux, a

white precipitate was formed. The reaction mixture was cooled to

room temperature, and the precipitate was ﬁltered and washed with

methanol (30 mL) to remove any unreacted 1,3-propanesultone. The

3-(Ethyl(3-hydroxy-4-nitrosophenyl)amino)propane-1-sulfonic

Acid Hydrochloride (11b).⁷²The compound was prepared according

to general procedure A from 8 (2.0 g, 7.71 mmol). Brown hygroscopic

powder. The yield was not determined, and a crude residue was

directly used in the next step.

3,3′-((3-Hydroxy-4-nitrosophenyl)azanediyl)bis(propane-1-sul-

fonic acid) Hydrochloride (11c). The compound was prepared

according to general procedure A from 10 (2.10 g, 5.94 mmol).

Brown hygroscopic powder. The yield was not determined, and a

crude residue was directly used in the next step.

General Procedure B: Synthesis of Nile Red Derivatives.

Compounds 11a−c (1 equiv, 4.34 mmol) and variously substituted 1-

naphthol (1 equiv, 4.34 mmol) were charged to a ﬂask. DMF (20 mL)

was added and the reaction mixture was stirred and heated to 150 °C

for 4 h under a nitrogen atmosphere. DMF was evaporated under a

reduced pressure, and the residue was puriﬁed by silica gel column

chromatography. The obtained compound was further puriﬁed by

recrystallization from a suitable mixture of solvents.

1

material was vacuum-dried to give a white powder, 10.1 g (84%). H

NMR (300 MHz, DMSO-d₆): δ (ppm) 11.43 (bs, 1H), 10.16 (bs,

1H), 7.36 (t, J = 8.0 Hz, 1H), 7.20−6.61 (m, 3H), 3.80−3.21 (bm,

4H), 2.61 (t, J = 6.7 Hz, 2H), 1.77 (bs, 2H), 0.98 (t, J = 7.1 Hz, 3H).

The spectroscopic data are consistent with those reported in the

literature.⁷²

3-((3-Hydroxyphenyl)amino)propane-1-sulfonic Acid (9). The

compound was prepared according to a known procedure.⁸⁷

A

solution of 1,3-propanesultone (1.5 equiv, 7.30 g, 59.8 mmol) in n-

butanol (20 mL) was added to a solution of 3-aminophenol (4.32 g,

J

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9-(Diethylamino)-5H-benzo[a]phenoxazin-5-one (Nile red)

(12a). The compound was prepared according to general procedure

B from 11a (3.48 g, 15.1 mmol) and 1-naphthol (2.17 g, 15.1 mmol).

The crude material was puriﬁed by gradient dichloromethane/ethyl

acetate (10:1 to 2:1, v/v) column chromatography and then

recrystallized from dichloromethane/hexane on a rotary evaporator

chemical shifts of some aromatic peaks diﬀer by up to 1 ppm

compared to the reported data. H NMR (300 MHz, DMSO-d₆): δ

1

(ppm) 10.32 (s, 1H), 8.41 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 9.0 Hz,

1H), 7.45 (d, J = 2.6 Hz, 1H), 7.21 (dd, J = 8.7, 2.7 Hz, 1H), 6.80

(dd, J = 9.2, 2.7 Hz, 1H), 6.63 (d, J = 2.7 Hz, 1H), 6.25 (s, 1H), 3.48

(q, J = 7.0 Hz, 4H), 1.16 (t, J = 6.9 Hz, 6H).¹³C{¹H} NMR (75 MHz,

DMSO-d₆): δ (ppm) 181.8, 159.6, 151.3, 150.0, 145.9, 138.7, 132.9,

130.3, 125.7, 124.0, 123.3, 120.0, 110.0, 109.8, 104.5, 96.0, 44.3, 12.4.

HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₂₀H₁₉N₂O₃335.1390;

found 335.1391.

9-(Diethylamino)-4-ﬂuoro-5H-benzo[a]phenoxazin-5-one (12e).

The compound was prepared according to general procedure B from

11a (498 mg, 2.16 mmol) and 8-ﬂuoronaphthalene-1-ol (6, 365 mg,

2.16 mmol). The crude material was puriﬁed by gradient dichloro-

methane/ethyl acetate (10:1 to 2:1, v/v) column chromatography and

then recrystallized from dichloromethane/hexane on a rotary

evaporator. Yield 157 mg (22%). Black powder. Mp 202−206 °C.

¹H NMR (300 MHz, CDCl₃): δ (ppm) 8.48 (d, J = 8.0 Hz, 1H), 7.62

(td, J = 8.1, 4.9 Hz, 1H), 7.55 (d, J = 9.1 Hz, 1H), 7.33−7.23 (m,

1H), 6.64 (dd, J = 9.1, 2.7 Hz, 1H), 6.43 (d, J = 2.7 Hz, 1H), 6.27 (s,

1H), 3.46 (q, J = 7.1 Hz, 4H), 1.26 (t, J = 7.1 Hz, 6H). ¹⁹F NMR

(282 MHz, CDCl₃): δ (ppm) −114.22 (dd, J = 11.6, 4.8 Hz).

¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 182.3 (d, J_C−F= 1.6 Hz),

161.4 (d, ¹J_C−F= 263.1 Hz), 151.4, 151.1, 146.9, 139.1 (d, J_C−F= 4.0

Hz), 134.7 (d, J_C−F= 1.5 Hz), 132.0 (d, J_C−F= 10.0 Hz), 131.3, 125.0,

1

to provide dark green crystals. Yield 0.99 g (21%). H NMR (300

MHz, CDCl₃): δ (ppm) 8.66 (dd, J = 7.8, 0.9 Hz, 1H), 8.31 (dd, J =

7.7, 1.1 Hz, 1H), 7.72 (td, J = 7.6, 1.5 Hz, 1H), 7.65 (td, J = 7.5, 1.4

Hz, 1H), 7.62 (d, J = 9.1 Hz, 1H), 6.68 (dd, J = 9.1, 2.7 Hz, 1H), 6.48

(d, J = 2.7 Hz, 1H), 6.39 (s, 1H), 3.48 (q, J = 7.1 Hz, 4H), 1.27 (t, J =

7.1 Hz, 6H). ¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 183.8,

152.2, 150.9, 146.9, 140.0, 132.2, 131.9, 131.4, 131.2, 130.0, 125.8,

125.1, 123.9, 109.8, 105.8, 96.4, 45.2, 12.8. UV−vis (CH₃OH, c = 1 ×

10⁻⁵M): λ_max(ε) = 264 (42200), 305 (11200), 553 (42100) nm (dm³

mol⁻¹cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em)

= 640 nm, Φ_F= 0.51. The spectroscopic data are consistent with

those reported in the literature.⁸⁹

9-(Diethylamino)-2-hydroxy-5H-benzo[a]phenoxazin-5-one

(12b). The compound was prepared according to general procedure B

from 11a (1.00 g, 4.34 mmol) and 1,6-dihydroxynaphthalene (0.694

g, 4.34 mmol). The crude material was puriﬁed by gradient

dichloromethane/ethyl acetate (10:1 to 2:1, v/v) column chromatog-

raphy and then recrystallized from dichloromethane/methanol on a

rotary evaporator to provide a dark green powder. Yield 0.230 g

(16%). ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 10.36 (s, 1H), 7.96

(d, J = 8.6 Hz, 1H), 7.87 (d, J = 2.3 Hz, 1H), 7.55 (d, J = 9.0 Hz, 1H),

7.08 (dd, J = 8.6, 2.4 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.60 (s, 1H),

6.13 (s, 1H), 3.48 (q, J = 6.9 Hz, 4H), 1.16 (t, J = 7.0 Hz, 6H).

¹³C{¹H} NMR (75 MHz, DMSO-d₆): δ (ppm) 181.5, 160.5, 151.5,

120.2 (d, J_C−F= 4.8 Hz), 120.0 (d, J_C−F= 4.0 Hz), 118.0 (d, J_C−F

=

22.2 Hz), 109.9, 107.0, 96.4, 45.2, 12.8. FTIR (neat, cm⁻¹): 3074,

3056, 2969, 2927, 2867, 1623, 1604, 1586, 1491, 1432, 1354, 1297,

1284, 1252, 1241, 1105, 926, 859, 793, 759. HRMS (APCI⁺) m/z: [M

+ H]⁺calcd for C₂₀H₁₈FN₂O₂337.1347; found 337.1347. UV−vis

(CH₃OH, c ∼ 2 × 10⁻⁵M): λ_max(ε) = 263 (29400), 308 (8930), 560

(33 000) nm (dm³mol⁻¹cm⁻¹). Fluorescence (CH₃OH,

A(λ_max(exc)) < 0.1): λ_max(em) = 647 nm, Φ_F= 0.46.

150.6, 146.3, 138.7, 133.7, 130.7, 127.4, 123.8, 123.8, 118.3, 109.8,

108.1, 104.0, 96.0, 44.3, 12.4. HRMS (APCI⁺) m/z: [M + H]⁺calcd

for C₂₀H₁₉N₂O₃335.1390; found 335.1388. UV−vis (CH₃OH, c = 9

× 10⁻⁶M): λ_max(ε) = 266 (34000), 547 (42 100) nm (dm³mol⁻¹

cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em) = 637

nm, Φ_F= 0.50. The spectroscopic data are consistent with those

reported in the literature.⁸⁸

9-(Diethylamino)-3-methoxy-5H-benzo[a]phenoxazin-5-one

(15). The compound was synthesized according to a literature

procedure.⁹¹12d (207 mg, 619 μmol) and KOH (3.0 equiv, 104 mg,

1.86 mmol) were charged in a ﬂask. DMF (10 mL) was added under a

nitrogen atmosphere followed by the addition of CH₃I (6.0 equiv, 230

μL, 3.71 mmol). The reaction mixture was stirred at room

temperature for 40 min. The reaction progress was monitored by

TLC (dichloromethane/ethyl acetate, 2:1, v/v). The solvent was

evaporated, and the residue was puriﬁed by column chromatography

eluting with dichloromethane/ethyl acetate (5:1, v/v). The crude

product was recrystallized from dichloromethane/hexane on evapo-

rator to yield ﬁne orange-red crystals. Yield 149 mg (69%). Mp 194−

9-(Diethylamino)-3-ﬂuoro-5H-benzo[a]phenoxazin-5-one (12c).

The compound was prepared according to general procedure B from

11a (435 mg, 1.89 mmol) and 7-ﬂuoronaphthalene-1-ol (2, 306 mg,

1.89 mmol). The crude material was puriﬁed by gradient dichloro-

methane/ethyl acetate (10:1 to 2:1, v/v) column chromatography and

then recrystallized from dichloromethane/hexane on a rotary

evaporator to provide dark green crystals. Yield 130 mg (21%). Mp

1

214−218 °C. H NMR (300 MHz, CDCl₃): δ (ppm) 8.66 (dd, J =

1

8.8, 5.3 Hz, 1H), 7.94 (dd, J = 9.3, 2.7 Hz, 1H), 7.61 (d, J = 9.1 Hz,

1H), 7.46−7.33 (m, 1H), 6.69 (dd, J = 9.1, 2.7 Hz, 1H), 6.49 (d, J =

2.7 Hz, 1H), 6.41 (s, 1H), 3.48 (q, J = 7.1 Hz, 4H), 1.27 (t, J = 7.1

Hz, 6H). ¹⁹F NMR (282 MHz, CDCl₃): δ (ppm) −109.03 (td, J =

8.7, 5.3 Hz). ¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 182.5 (d,

197 °C. H NMR (300 MHz, CDCl₃): δ (ppm) 8.56 (d, J = 8.8 Hz,

1H), 7.74 (d, J = 2.8 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.26 (dd, J =

8.8, 2.7 Hz, 1H), 6.67 (dd, J = 9.1, 2.7 Hz, 1H), 6.48 (d, J = 2.7 Hz,

1H), 6.39 (s, 1H), 3.96 (s, 3H), 3.46 (q, J = 7.1 Hz, 4H), 1.26 (t, J =

7.1 Hz, 6H). ¹³C{¹H} NMR (75 MHz, CDCl₃): δ (ppm) 183.5,

161.6, 151.9, 150.4, 146.6, 140.0, 133.6, 130.9, 125.9, 125.5, 125.2,

120.4, 109.8, 107.2, 105.7, 96.5, 55.8, 45.2, 12.8. FTIR (neat, cm⁻¹):

3072, 3003, 2972, 2929, 2867, 2835, 1621, 1588, 1564, 1515, 1494,

1405, 1343, 1310, 1259, 1115, 1066, 1017, 851, 830, 794, 622. HRMS

(APCI⁺) m/z: [M + H]⁺calcd for C₂₁H₂₁N₂O₃349.1547; found

349.1547. UV−vis (CH₃OH, c ∼ 9.0 × 10⁻⁶M): λ_max(ε) = 266

(39400), 300 (8180), 552 (43100) nm (dm³mol⁻¹cm⁻¹).

J

_C−F= 2.1 Hz), 164.2 (d, ¹J_C−F= 251.5 Hz), 152.3, 151.0, 146.9, 139.1

(d, J_C−F= 1.4 Hz), 134.1 (d, J_C−F= 7.5 Hz), 131.2, 128.5 (d, J_C−F

=

2.8 Hz), 126.7 (d, J_C−F= 8.3 Hz), 125.2, 119.3 (d, J_C−F= 23.3 Hz),

111.7 (d, J_C−F= 22.9 Hz), 110.1, 105.8, 96.4, 45.3, 12.8. FTIR (neat,

cm⁻¹): 3063, 2969, 2926, 1623, 1601, 1584, 1563, 1515, 1488, 1448,

1403, 1320, 1277, 1245, 1179, 1099, 1077, 1053, 1017, 922, 845, 830,

799, 619. HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₂₀H₁₈FN₂O₂

337.1347; found 337.1348. UV−vis (CH₃OH, c ∼ 9.5 × 10⁻⁵M):

λ_max(ε) = 266 (10200), 304 (2470), 557 (11700) nm (dm³mol⁻¹

cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em) = 643

nm, Φ_F= 0.44.

9-(Diethylamino)-3-hydroxy-5H-benzo[a]phenoxazin-5-one

(12d). The compound was prepared according to general procedure B

from 11a (1.17 g, 5.05 mmol) and 1,7-dihydroxynaphthalene (0.809

g, 5.05 mmol). The crude material was puriﬁed by gradient

dichloromethane/ethyl acetate (10:1 to 2:1, v/v) column chromatog-

raphy and then recrystallized from dichloromethane/methanol on a

rotary evaporator to provide a ﬁne green powder. Yield 0.370 g

(26%). The number of peaks and coupling constants (¹H NMR) are

consistent with those reported in the literature;⁹⁰however, the

3-(Ethyl(5-oxo-5H-benzo[a]phenoxazin-9-yl)amino)propane-1-

sulfonic Acid (13a). The compound was prepared according to

general procedure B from 11b (2.22 g, 7.70 mmol) and 1-naphthol

(1.11 g, 7.70 mmol). The crude material was puriﬁed by column

chromatography using ethyl acetate/i-propanol/H₂O (4:2:1, v/v) as a

mobile phase and then recrystallized from methanol/ethyl acetate on

a rotary evaporator. Yield 0.740 g (28%). Green powder. Mp 191−

1

194 °C. H NMR (300 MHz, DMSO-d₆): δ (ppm) 8.58 (d, J = 7.8

Hz, 1H), 8.13 (d, J = 7.4 Hz, 1H), 7.93−7.76 (m, 1H), 7.76−7.67 (m,

1H), 7.62 (d, J = 9.0 Hz, 1H), 6.91 (dd, J = 9.1, 2.6 Hz, 1H), 6.73 (d,

J = 2.5 Hz, 1H), 6.30 (s, 1H), 3.65−3.45 (m, 4H), 2.55−2.51 (m, 2H,

overlap with solvent peak), 1.89 (p, J = 6.9 Hz, 2H), 1.16 (t, J = 6.9

Hz, 3H). ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ (ppm) 181.9,

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151.8, 151.1, 146.3, 138.2, 131.6, 131.5, 131.0, 130.8, 129.8, 125.0,

124.2, 123.3, 110.5, 104.5, 96.1, 49.2, 48.4, 44.8, 23.4, 12.2. FTIR

(neat, cm⁻¹): 3442 (broad), 3064, 2970, 2929, 2891, 1617, 1581,

1555, 1515, 1490, 1459, 1408, 1366, 1347, 1309, 1272, 1253, 1224,

1179, 1132, 1110, 1077, 1046, 1004, 777. HRMS (ESI⁻) m/z: [M −

H]⁻calcd for C₂₁H₁₉N₂O₅S 411.1020; found 411.1021. UV−vis

(CH₃OH, c ∼ 1.6 × 10⁻⁵M): λ_max(ε) = 264 (29100), 552 (30400)

nm (dm³mol⁻¹cm⁻¹). UV−vis (PBS 10 mM, I = 0.1 M, c ∼ 1.6 ×

10⁻⁵M): λ_max(ε) = 268 (33100), 564 (32900) nm (dm³mol⁻¹cm⁻¹).

Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em) = 640 nm, Φ_F

= 0.44. Fluorescence (PBS 10 mM, I = 0.1 M, A(λ_max(exc)) < 0.1):

λ_max(em) = 659 nm, Φ_F= 0.063.

3-(Ethyl(2-hydroxy-5-oxo-5H-benzo[a]phenoxazin-9-yl)amino)-

propane-1-sulfonic Acid (13b). The compound was prepared

according to general procedure B from 11b (2.22 g, 7.70 mmol)

and 1,6-dihydroxynaphthalene (1.23 g, 7.70 mmol). The crude

material was puriﬁed by column chromatography using ethyl acetate/

i-propanol/H₂O (2:2:1, v/v) as a mobile phase and then recrystallized

from methanol/ethyl acetate on a rotary evaporator. Yield 0.560 g

(17%). Dark violet powder. ¹H NMR (500 MHz, DMSO-d₆): δ

(ppm) 10.38 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 2.5 Hz,

1H), 7.56 (d, J = 9.0 Hz, 1H), 7.08 (dd, J = 8.6, 2.5 Hz, 1H), 6.86

(dd, J = 9.1, 2.7 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 6.14 (s, 1H), 3.55

(t, J = 7.9 Hz, 2H), 3.51 (q, J = 7.0 Hz, 2H), 2.55−2.49 (m, 2H,

overlap with solvent peak), 1.89 (p, J = 7.3 Hz, 2H), 1.15 (t, J = 7.0

Hz, 3H). ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ (ppm) 181.5,

160.5, 151.6, 151.0, 146.3, 138.6, 133.7, 130.7, 127.4, 123.9, 123.8,

118.3, 110.1, 108.1, 104.0, 96.2, 49.2, 48.4, 44.8, 23.4, 12.2. FTIR

(neat, cm⁻¹): 3406 (broad), 2973, 2931, 1641, 1588, 1561, 1518,

1481, 1441, 1410, 1319, 1272, 1181, 1119, 1089, 1043, 910, 824.

HRMS (ESI⁻) m/z: [M − H]⁻calcd for C₂₁H₁₉N₂O₆S 427.0969;

found 427.0971. UV−vis (CH₃OH, c ∼ 1.5 × 10⁻⁵M): λ_max(ε) = 264

(29 000), 549 (29800) nm (dm³mol⁻¹cm⁻¹). UV−vis (PBS 10 mM,

I = 0.1 M, c ∼ 1.5 × 10⁻⁵M): λ_max(ε) = 262 (27200), 560 (25800)

nm (dm³mol⁻¹cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1):

λ_max(em) = 637 nm, Φ_F= 0.54. Fluorescence (PBS 10 mM, I = 0.1 M,

A(λ_max(exc)) < 0.1): λ_max(em) = 655 nm, Φ_F= 0.097. The

spectroscopic data are in agreement with those reported in the

literature.⁹²

3,3′-((5-Oxo-5H-benzo[a]phenoxazin-9-yl)azanediyl)bis-

(propane-1-sulfonic Acid) (14a). The compound was prepared

according to general procedure B from 11c (2.27 g, 7.87 mmol) and

1-naphthol (1.14 g, 7.87 mmol). The crude material was puriﬁed by

column chromatography using a gradient of ethyl acetate/i-propanol/

H₂O (4:2:1 to 1:1:1, v/v) as a mobile phase and then recrystallized

from methanol/ethyl acetate on a rotary evaporator. Yield 0.175 g

(5%). Dark violet powder. Mp > 220 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ (ppm) 8.58 (dd, J = 8.0, 1.3 Hz, 1H), 8.13 (dd, J = 7.8,

1.4 Hz, 1H), 7.81 (td, J = 7.6, 1.5 Hz, 1H), 7.71 (td, J = 7.5, 1.4 Hz,

1H), 7.62 (d, J = 9.1 Hz, 1H), 6.97 (dd, J = 9.2, 2.6 Hz, 1H), 6.79 (d,

J = 2.5 Hz, 1H), 6.31 (s, 1H), 3.57 (t, J = 7.8 Hz, 4H), 2.57−2.49 (m,

4H, overlap with solvent peak), 1.89 (p, J = 7.3 Hz, 4H). ¹³C{¹H}

NMR (75 MHz, DMSO-d₆): δ (ppm) 181.9, 151.9, 146.4, 131.6,

131.5, 130.8, 129.8, 127.5, 125.0, 124.3, 123.4, 123.4, 110.7, 104.5,

96.3, 92.9, 49.6, 48.4, 23.2. FTIR (neat, cm⁻¹): 3432, 3067, 2941,

2883, 1617, 1585, 1557, 1492, 1461, 1412, 1366, 1313, 1183, 1137,

1116, 1048, 1005. HRMS (ESI⁻) m/z: [M − H]⁻calcd for

C₂₂H₂₁N₂O₈S₂505.0745; found 505.0745. UV−vis (CH₃OH, c ∼ 1.3

× 10⁻⁵M): λ_max(ε) = 264 (24700), 552 (24500) nm (dm³mol⁻¹

cm⁻¹). UV−vis (PBS 10 mM, I = 0.1 M, c ∼ 1.3 × 10⁻⁵M): λ_max(ε) =

267 (24800), 573 (25900) nm (dm³mol⁻¹cm⁻¹). Fluorescence

(CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em) = 640 nm, Φ_F= 0.55.

Fluorescence (PBS 10 mM, I = 0.1 M, A(λ_max(exc)) < 0.1): λ_max(em)

= 654 nm, Φ_F= 0.19.

from methanol/ethyl acetate on a rotary evaporator. Yield 0.200 g

(8%). Dark violet powder. Mp > 220 °C. ¹H NMR (500 MHz,

DMSO-d₆): δ (ppm) 10.39 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.91 (d,

J = 2.5 Hz, 1H), 7.55 (d, J = 9.1 Hz, 1H), 7.08 (dd, J = 8.6, 2.5 Hz,

1H), 6.92 (dd, J = 9.1, 2.7 Hz, 1H), 6.74 (d, J = 2.6 Hz, 1H), 6.15 (s,

1H), 3.56 (t, J = 7.8 Hz, 4H), 2.54−2.51 (m, 4H, overlap with solvent

peak), 1.88 (p, J = 7.4 Hz, 4H). ¹³C{¹H} NMR (126 MHz, DMSO-

d₆): δ (ppm) 181.5, 160.5, 151.6, 151.2, 146.3, 138.6, 133.7, 130.6,

127.4, 123.9, 118.3, 110.3, 108.1, 104.0, 99.5, 96.3, 49.5, 48.4, 23.2.

FTIR (neat, cm⁻¹): 3443, 3249, 2932, 2884, 1622, 1590, 1562, 1516,

1470, 1409, 1321, 1166, 1118, 1042, 911, 821, 741, 587, 521. HRMS

(ESI⁻) m/z: [M − H]⁻calcd for C₂₂H₂₁N₂O₉S₂521.0694; found

521.0696. UV−vis (CH₃OH, c ∼ 1.3 × 10⁻⁵M): λ_max(ε) = 262

(21300), 546 (20200) nm (dm³mol⁻¹cm⁻¹). UV−vis (PBS 10 mM,

I = 0.1 M, c ∼ 1.3 × 10⁻⁵M): λ_max(ε) = 261 (30600), 561 (27400)

nm (dm³mol⁻¹cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1):

λ_max(em) = 637 nm, Φ_F= 0.60. Fluorescence (PBS 10 mM, I = 0.1 M,

A(λ_max(exc)) < 0.1): λ_max(em) = 652 nm, Φ_F= 0.13.

General Procedure C: Synthesis of Pd Sensors. This general

procedure was optimized by modiﬁcation of literature proce-

dures.^40,55,56Nile red derivative (12−15, 1.0 equiv, 400 μmol) and

recrystallized Pd(OAc)₂(1.0−2.0 equiv) were placed in a ﬂask, and

acetic acid (20 mL) was added under a nitrogen atmosphere. The

mixture was stirred for 16 h at 65 °C. Acetic acid was evaporated

under reduced pressure, and the residue was recrystallized from a

suitable mixture of solvents on a rotary evaporator (the solution was

ﬁltered through a pad of Celite prior to recrystallization) to provide a

Pd complex as a dark violet-blue powder.

[(9-(Diethylamino)-5H-benzo[a]phenoxazin-5-one)Pd(μ-OAc)]₂

(16a). The compound was prepared according to general procedure C

from 12a (141 mg, 443 μmol) and Pd(OAc)₂(114 mg, 508 μmol).

The residue was recrystallized from dichloromethane/hexane and

then washed with diethyl ether (3 × 10 mL) on the frit to remove

unreacted starting material. The title compound is insoluble in diethyl

ether and hexane; on the other hand, starting material is soluble in

both dichloromethane and diethyl ether. Yield 150 mg (70%). Dark

blue powder. ¹H NMR (500 MHz, CDCl₃): δ (ppm) 7.71 (d, J = 9.2

Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 6.77 (d, J = 7.7 Hz, 2H), 6.59 (t, J

= 7.6 Hz, 2H), 6.33 (dd, J = 9.4, 2.6 Hz, 2H), 6.17 (d, J = 2.7 Hz,

2H), 6.04 (s, 2H), 3.46 (q, J = 6.9 Hz, 8H), 2.28 (s, 6H), 1.29 (t, J =

7.1 Hz, 12H). ¹³C{¹H} NMR (126 MHz, CDCl₃): δ (ppm) 184.5,

181.8, 150.4, 150.4, 149.1, 148.4, 147.3, 142.3, 133.9, 130.7, 128.4,

127.9, 123.2, 121.3, 109.2, 107.4, 97.2, 45.3, 25.1, 12.9. FTIR (neat,

cm⁻¹): 3061, 2970, 2928, 2868, 1634, 1616, 1570, 1545, 1519, 1489,

1405, 1380, 1351, 1302, 1283, 1250, 1187, 1120, 1094, 1077, 1019,

846, 795, 770. HRMS (APCI⁺) m/z: [M + H]⁺calcd for

C₄₄H₄₁N₄O₈Pd₂967.1012; found 967.1060. UV−vis (CH₃OH/

dichloromethane (9:1), c ∼ 1 × 10⁻⁵M): λ_max(ε) = 610 (37900)

nm (dm³mol⁻¹cm⁻¹). Fluorescence (CH₃OH/dichloromethane

(9:1), A(λ_max(exc)) < 0.1): λ_max(em) = 715 nm, Φ_F= 0.080. The

spectroscopic data are consistent with those reported in the

literature.^40,55,56

[(9-(Diethylamino)-2-hydroxy-5H-benzo[a]phenoxazin-5-one)-

Pd(μ-OAc)]₂(16b). The compound was prepared according to general

procedure C from 12b (57.6 mg, 172 μmol) and Pd(OAc)₂(65.0 mg,

290 μmol). The residue was recrystallized from dichloromethane/

hexane and then washed with diethyl ether (3 × 10 mL) on the frit.

Yield 80 mg (93%). Dark blue powder. Mp > 220 °C. ¹H NMR (500

MHz, CDCl₃): δ (ppm) 8.18 (s, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.34

(d, J = 9.4 Hz, 2H), 6.27 (dd, J = 9.5, 2.7 Hz, 2H), 6.11−5.98 (m,

4H), 5.90 (s, 2H), 3.42 (q, J = 7.2 Hz, 8H), 2.19 (s, 6H), 1.26 (t, J =

7.1 Hz, 12H). ¹³C{¹H} NMR (126 MHz, CDCl₃): δ (ppm) 183.5,

183.2, 163.8, 150.4, 150.1, 149.6, 146.8, 143.8, 128.3, 126.7, 124.5,

124.2, 122.7, 117.6, 109.1, 107.7, 97.4, 45.3, 24.1, 12.9. FTIR (neat,

cm⁻¹): 3282, 2973, 2929, 2873, 1617, 1583, 1565, 1520, 1494, 1399,

1342, 1311, 1269, 1184, 1114, 1098, 1075, 929, 826, 795. HRMS

(APCI⁺) m/z: [M + H]⁺calcd for C₂₂H₂₁N₂O₅Pd 499.0489; found

499.0492, dimer not observed. UV−vis (CH₃OH/dichloromethane

(9:1), c ∼ 1 × 10⁻⁵M): λ_max(ε) = 610 (33500) nm (dm³mol⁻¹

3,3′-((2-Hydroxy-5-oxo-5H-benzo[a]phenoxazin-9-yl)azanediyl)-

bis(propane-1-sulfonic Acid) (14b). The compound was prepared

according to general procedure B from 11c (1.80 g, 4.71 mmol) and

1,6-dihydroxynaphthalene (0.754 g, 4.71 mmol). The crude material

was puriﬁed by column chromatography using ethyl acetate/i-

propanol/H₂O (1:1:2 v/v) as a mobile phase and then recrystallized

L

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cm⁻¹). Fluorescence (CH₃OH/dichloromethane (9:1), A(λ_max(exc))

< 0.1): λ_max(em) = 718 nm, Φ_F= 0.010.

μmol) and Pd(OAc)₂(84.0 mg, 374 μmol). The residue was

sonicated ﬁrst in dichloromethane (10 mL), then in methanol (10

mL), and then ﬁltered. The solid was dissolved in water (10 mL),

then ﬁltered through a pad of Celite, and the solvent was evaporated.

We were not able to interpret the NMR spectra because of a strong

broadening of peaks (in diﬀerent solvents and at diﬀerent temper-

[(9-(Diethylamino)-3-ﬂuoro-5H-benzo[a]phenoxazin-5-one)Pd-

(μ-OAc)]₂(16c). The compound was prepared according to general

procedure C from 12c (134 mg, 398 μmol) and Pd(OAc)₂(100 mg,

445 μmol). The residue was recrystallized from dichloromethane/

hexane and then washed with diethyl ether (3 × 10 mL) on the frit.

Yield 82 mg (41%). Dark violet-blue powder. ¹³C was not possible to

resolve because of a low solubility of the compound in CD₂Cl₂and

splitting of the signals by ﬂuorine. Mp > 220 °C. ¹H NMR (500 MHz,

CD₂Cl₂): δ (ppm) 7.68 (d, J = 9.3 Hz, 2H), 7.01 (dd, J = 9.1, 1.9 Hz,

2H), 6.47 (dd, J = 9.4, 2.8 Hz, 2H), 6.41 (dd, J = 8.5, 1.9 Hz, 2H),

6.23 (d, J = 2.8 Hz, 2H), 5.95 (s, 2H), 3.48 (q, J = 7.3 Hz, 8H), 2.27

(s, 6H), 1.29 (t, J = 7.2 Hz, 12H). ¹⁹F NMR (471 MHz, CD₂Cl₂): δ

(ppm) −104.51 (s). FTIR (neat, cm⁻¹): 3070, 2974, 2928, 2870,

1634, 1617, 1577, 1548, 1520, 1492, 1439, 1408, 1384, 1349, 1316,

1282, 1240, 1183, 1118, 1078, 1060, 840, 796. HRMS (APCI⁺) m/z:

[M + H]⁺calcd for C₄₄H₃₉F₂N₄O₈Pd₂1003.0824; found 1003.0837.

UV−vis (CH₃OH/dichloromethane (9:1), c ∼ 1 × 10⁻⁵M): λ_max(ε)

= 630 (32900) nm (dm³mol⁻¹cm⁻¹). Fluorescence (CH₃OH/

dichloromethane (9:1), A(λ_max(exc)) < 0.1): λ_max(em) = 717 nm, Φ_F

= 0.065.

1

atures). Yield 40 mg (60%). Dark blue powder. Mp > 220 °C. H

NMR (500 MHz, D₂O, a strong signal broadening): δ 7.09, 6.58,

6.49, 6.39, 6.22, 5.77, 3.61, 3.09, 3.07, 3.06, 2.23, 2.21, 2.19, 2.17,

2.16, 2.16. FTIR (neat, cm⁻¹): 3412 (broad), 2927, 1634, 1567, 1540,

1488, 1404, 1367, 1307, 1278, 1163, 1123, 1038, 797. HRMS (ESI⁻)

m/z: [M − 2H]²⁻calcd for C₂₄H₂₂N₂O₁₀PdS₂333.9885; found

333.9890, dimer not observed.

[(3,3′-((2-Hydroxy-5-oxo-5H-benzo[a]phenoxazin-9-yl)-

azanediyl)bis(propane-1-sulfonic acid))Pd(μ-OAc)]₂(18b). The

compound was prepared according to general procedure C from

14b (73.0 mg, 140 μmol) and Pd(OAc)₂(138 mg, 615 μmol). The

residue was sonicated ﬁrst in dichloromethane (10 mL), then in

methanol (10 mL), and then ﬁltered. The solid was dissolved in water

(10 mL), then ﬁltered through a pad of Celite, and solvent was

evaporated. We were not able to interpret the NMR spectra because

of a strong broadening of peaks (in diﬀerent solvents and at diﬀerent

temperatures). Mp > 220 °C. NMR (300 MHz, DMSO-d₆): a strong

signal broadening. FTIR (neat, cm⁻¹): 3421, 2930, 1634, 1575, 1402,

1317, 1171, 1120, 1039. HRMS (ESI⁻) m/z: [M − H]⁻calcd for

C₂₄H₂₃N₂O₁₁PdS₂684.9784, found 684.9771, dimer not observed.

Synthesis of Triﬂuoroacetate Complex 20. [(9-(Diethylami-

no)-5H-benzo[a]phenoxazin-5-one)Pd(μ-OOCCF₃)]₂(20). Nile red

(12a, 50.0 mg, 157 μmol) and recrystallized Pd(OAc)₂(1.2 equiv,

42.3 mg, 188 μmol) were charged in a ﬂask, and triﬂuoroacetic acid

(10 mL) was added. The mixture was stirred under a nitrogen

atmosphere for 16 h at 65 °C. After cooling down to room

temperature, a suﬃcient amount of diethyl ether (100 mL) was

added, which caused precipitation. A dark blue-green solid was

ﬁltered, and the crude material (triﬂuoroacetate salt of the title

compound) was redissolved in acetone. Anhydrous K₂CO₃(∼0.5 g)

was added to the solution, and the suspension was stirred for 1 h at

room temperature. Acetone was evaporated, and the residue was

redissolved in dichloromethane (50 mL) and ﬁltered. The same

volume of hexane was added to the mixture, and the major part of

dichloromethane was evaporated under reduced pressure, which

caused precipitation. The solid was ﬁltered and washed with diethyl

ether (2 × 10 mL) and hexane (2 × 10 mL). The residue was dried

under vacuum. Yield 40 mg (47%). Dark blue solid. Mp > 220 °C. ¹H

NMR (300 MHz, CD₂Cl₂): δ (ppm) 7.33 (d, J = 7.3 Hz, 2H), 7.29

(d, J = 9.3 Hz, 2H), 6.60−6.42 (m, 4H), 6.39 (dd, J = 9.4, 2.7 Hz,

2H), 6.17 (d, J = 2.7 Hz, 2H), 5.80 (s, 2H), 3.47 (q, J = 7.2 Hz, 8H),

1.27 (t, J = 7.1 Hz, 12H). ¹⁹F NMR (471 MHz, CDCl₃): δ (ppm)

-74.62 (s). ¹³C{¹H} NMR (126 MHz, CDCl₃): δ (ppm) 183.7, 165.9

[(9-(Diethylamino)-3-methoxy-5H-benzo[a]phenoxazin-5-one)-

Pd(μ-OAc)]₂(19). The compound was prepared according to general

procedure C from 15 (55.0 mg, 158 μmol) and Pd(OAc)₂(69.0 mg,

307 μmol). The residue was recrystallized from dichloromethane/

hexane and then washed with diethyl ether (3 × 10 mL) on the frit.

Yield 60 mg (74%). Dark blue powder. ¹H NMR (500 MHz, CDCl₃):

δ (ppm) 7.72 (d, J = 9.3 Hz, 2H), 6.86 (d, J = 2.3 Hz, 2H), 6.40 (dd, J

= 9.4, 2.8 Hz, 2H), 6.28 (d, J = 2.3 Hz, 2H), 6.22 (d, J = 2.7 Hz, 2H),

6.00 (s, 2H), 3.54 (s, 6H, OCH₃), 3.44 (q, J = 7.1 Hz, 8H), 2.27 (s,

6H), 1.27 (t, J = 7.2 Hz, 12H). ¹³C{¹H} NMR (126 MHz, CDCl₃): δ

(ppm) 184.3, 181.7, 159.4, 150.38, 150.37, 149.9, 148.3, 146.9, 135.8,

131.7, 127.7, 122.9, 120.7, 109.0, 107.1, 105.2, 97.0, 55.1, 45.0, 25.1,

12.8. HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₄₆H₄₅N₄O₁₀Pd₂

1027.1224; found 1027.1216.

[(3-(Ethyl(5-oxo-5H-benzo[a]phenoxazin-9-yl)amino)propane-1-

sulfonic acid)Pd(μ-OAc)]₂(17a). The compound was prepared

according to general procedure C from 13a (150 mg, 364 μmol)

and Pd(OAc)₂(101 mg, 450 μmol). The residue was sonicated in

dichloromethane (10 mL) and then ﬁltered. The solid was dissolved

in methanol (50 mL) and then ﬁltered through a pad of Celite, and

title compound was obtained by addition of dichloromethane (100

mL) to this solution and ﬁltering of precipitated solids. We were not

able to interpret the NMR spectra because of a strong broadening of

peaks (in diﬀerent solvents and at diﬀerent temperatures). Yield 60

1

mg (29%). Dark blue powder. Mp > 220 °C. H NMR (300 MHz,

CD₃OD, a strong signal broadening): δ (ppm) 7.59, 7.25, 6.62, 6.28,

5.83, 5.48, 3.62, 3.55, 2.93, 2.16, 1.28, 1.28. FTIR (neat, cm⁻¹): 3412

(broad), 2970, 2929, 1627, 1571, 1538, 1488, 1404, 1277, 1128, 1037.

HRMS (ESI⁻) m/z: [M − H]⁻calcd for C₂₃H₂₁N₂O₇PdS 575.0118;

found 575.0082, a larger error is caused by averaging of the signals of

monomer and dimer (see Figure S72).

2

(q, J_C−F= 38.7 Hz), 150.8, 149.8, 147.6, 147.1, 146.5, 141.8, 132.4,

1

130.7, 128.0, 127.5, 122.5, 121.9, 115.4 (q, J_C−F= 287.4 Hz), 109.5,

107.4, 97.3, 45.4, 12.8. FTIR (neat, cm⁻¹): 3066, 2972, 2929, 2872,

1659, 1634, 1616, 1574, 1546, 1520, 1491, 1407, 1347, 1279, 1196,

1184, 1117, 1091, 1075, 1018, 846, 795, 767, 730. HRMS (APCI⁺)

m/z: [M + H]⁺calcd for C₄₄H₃₅F₆N₄O₈Pd₂1075.0447; found

1075.0462. UV−vis (CH₃OH/dichloromethane (9:1), c ∼ 1 × 10⁻⁵

M): λ_max(ε) = 616 (24500) nm (dm³mol⁻¹cm⁻¹). Fluorescence

(CH₃OH/dichloromethane (9:1), A(λ_max(exc)) < 0.1): λ_max(em) =

716 nm, Φ_F= 0.080.

[(3-(Ethyl(2-hydroxy-5-oxo-5H-benzo[a]phenoxazin-9-yl)-

amino)propane-1-sulfonic acid)Pd(μ-OAc)]₂(17b). The compound

was prepared according to general procedure C from 13b (100 mg,

364 μmol) and Pd(OAc)₂(130 mg, 579 μmol). The residue was

sonicated in dichloromethane (10 mL) and then ﬁltered. The solid

was dissolved in methanol (50 mL) and then ﬁltered through a pad of

Celite, and the title compound was obtained by the addition of

dichloromethane (100 mL) to this solution and ﬁltering of the

precipitated solids. We were not able to interpret the NMR spectra

because of a strong broadening of peaks (in diﬀerent solvents and at

diﬀerent temperatures). Yield 66 mg (48%). Dark blue powder. Mp >

220 °C. NMR (300 MHz, DMSO-d₆): a strong signal broadening.

HRMS (ESI⁻) m/z: [M − H]⁻calcd for C₂₃H₂₁N₂O₈PdS 591.0068;

found 591.0064, dimer not observed.

General Procedure D: Preparative Synthesis of Methyl Ester

Derivatives. A solution of palladacycle (16a−c, 19, 7 μmol) in

methanol (10 mL) was stirred under CO atmosphere for 16 h at room

temperature. The black precipitate formed during the reaction (Pd

black) was separated. The mixture was then ﬁltered through a pad of

Celite, and the ﬁltrate was evaporated. The residue was puriﬁed by

silica gel column chromatography to aﬀord the methyl ester.

Methyl 9-(Diethylamino)-5-oxo-5H-benzo[a]phenoxazine-1-car-

boxylate (21a). The compound was prepared according to general

procedure D from 16a (200 mg, 207 μmol) and methanol (60 mL).

Mobile-phase dichloromethane/ethyl acetate (2:1, v/v) was used for

[(3,3′-((5-Oxo-5H-benzo[a]phenoxazin-9-yl)azanediyl)bis-

(propane-1-sulfonic acid))Pd(μ-OAc)]₂(18a). The compound was

prepared according to general procedure C from 14a (50.0 mg, 98.7

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J. Org. Chem. XXXX, XXX, XXX−XXX

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1

puriﬁcation. Yield 70 mg (45%). Black powder. Mp 184−188 °C. H

NMR (500 MHz, CDCl₃): δ (ppm) 8.41 (dd, J = 6.7, 2.6 Hz, 1H),

7.69−7.62 (m, 2H), 7.47 (d, J = 9.2 Hz, 1H), 6.65 (dd, J = 9.1, 2.7

Hz, 1H), 6.47 (d, J = 2.7 Hz, 1H), 6.40 (s, 1H), 4.00 (s, 3H), 3.46 (q,

J = 7.1 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). ¹³C{¹H} NMR (126 MHz,

CDCl₃): δ (ppm) 182.8, 171.4, 152.4, 151.4, 146.9, 138.1, 132.5,

131.6, 131.3, 130.4, 129.4, 128.7, 127.6, 124.5, 110.2, 105.7, 96.5,

52.4, 45.4, 12.7. FTIR (neat, cm⁻¹): 2963, 2945, 2924, 2869, 1733,

1615, 1581, 1562, 1489, 1468, 1414, 1358, 1311, 1268, 1242, 1179,

1113, 1070, 1017, 812, 774. HRMS (APCI⁺) m/z: [M + H]⁺calcd for

C₂₂H₂₁N₂O₄377.1496; found 377.1496. UV−vis (CH₃OH, c ∼ 1.3 ×

10⁻⁵M): λ_max(ε) = 268 (32100), 315 (7420), 561 (40100) nm (dm³

mol⁻¹cm⁻¹). Fluorescence (CH₃OH, A(λ_max(exc)) < 0.1): λ_max(em)

= 635 nm, Φ_F= 0.54.

Methyl 9-(Diethylamino)-2-hydroxy-5-oxo-5H-benzo[a]-

phenoxazine-1-carboxylate (21b). The compound was prepared

according to general procedure D from 16b (7.0 mg, 7.02 μmol).

Dichloromethane/ethyl acetate (1:1, v/v) was used as a mobile phase.

Yield was not determined (only an analytical amount was obtained).

Dark violet solid. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 10.82 (s,

1H), 8.07 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 9.1 Hz, 1H), 7.23 (d, J =

8.7 Hz, 1H), 6.82 (dd, J = 9.2, 2.7 Hz, 1H), 6.64 (d, J = 2.6 Hz, 1H),

6.19 (s, 1H), 3.89 (s, 3H), 3.50 (q, J = 7.0 Hz, 4H), 1.16 (t, J = 6.8

Hz, 6H). HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₂₂H₂₁N₂O₅

393.1445; found 393.1445.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.joc.9b03217.

■

sı

NMR and optical spectra; HRMS, ESI-MS, SVD, and

target analysis data; stability measurements; and

ﬂuorescence microscopy data (PDF)

AUTHOR INFORMATION

Corresponding Author

■

́

Petr Klan − Department of Chemistry and RECETOX, Faculty

of Science, Masaryk University, 625 00 Brno, Czech Republic;

orcid.org/0000-0001-6287-2742; Phone: +420-54949-

4856; Email: klan@sci.muni.cz; Fax: +420-54949-2443

Authors

Dominik Madea − Department of Chemistry and RECETOX,

Faculty of Science, Masaryk University, 625 00 Brno, Czech

Republic

Marek Martínek − Department of Chemistry and RECETOX,

Faculty of Science, Masaryk University, 625 00 Brno, Czech

Republic

́

Lucie Muchova − Institute of Medical Biochemistry and

Methyl 9-(Diethylamino)-3-ﬂuoro-5-oxo-5H-benzo[a]-

phenoxazine-1-carboxylate (21c). The compound was prepared

according to general procedure D from 16c (5.6 mg, 5.59 μmol).

Dichloromethane/ethyl acetate (10:1, v/v) was used as a mobile

phase. Yield was not determined (only an analytical amount was

Laboratory Diagnostics, 1st Faculty of Medicine, Charles

University, 121 08 Praha 2, Czech Republic

̌

́

̌

Jirí Vana − Institute of Organic Chemistry and Technology,

Faculty of Chemical Technology, University of Pardubice, 532

10 Pardubice, Czech Republic

1

obtained). Dark black solid. H NMR (300 MHz, CDCl₃): δ (ppm)

Libor Vítek − Institute of Medical Biochemistry and Laboratory

Diagnostics, 1st Faculty of Medicine, Charles University, 121 08

Praha 2, Czech Republic

8.07 (dd, J = 8.8, 2.8 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.37 (dd, J =

7.8, 2.8 Hz, 1H), 6.68 (dd, J = 9.1, 2.7 Hz, 1H), 6.49 (d, J = 2.7 Hz,

1H), 6.42 (s, 1H), 4.01 (s, 3H), 3.48 (q, J = 7.1 Hz, 4H), 1.27 (t, J =

7.1 Hz, 6H). ¹⁹F NMR (282 MHz, CDCl₃): δ (ppm) -108.51 (t, J =

8.3 Hz). HRMS (APCI⁺) m/z: [M + H]⁺calcd for C₂₂H₂₀FN₂O₄

395.1402; found 395.1402.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.joc.9b03217

Methyl 9-(Diethylamino)-3-methoxy-5-oxo-5H-benzo[a]-

phenoxazine-1-carboxylate (22). The compound was prepared

according to general procedure D from 19 (7.0 mg, 6.83 μmol).

The crude material was not puriﬁed by column chromatography.

Notes

The authors declare no competing ﬁnancial interest.

1

ACKNOWLEDGMENTS

Support for this work was provided by the Czech Science

Foundation (GA18-12477S). The authors thank Lubos Jılek

and Peter Sebej (Masaryk University) for their help with the

According to H NMR and HRMS, the product of the reaction is a

■

mixture of the title compound and 15 in the ratio of 1.4:1 (calculated

from integration of ¹H NMR signals). Yield was not determined (only

an analytical amount was obtained). Dark violet solid. ¹H NMR (title

compound, 300 MHz, CDCl₃): δ (ppm) 7.86 (d, J = 2.7 Hz, 1H),

7.46 (d, J = 9.1 Hz, 1H), 7.21 (d, J = 2.7 Hz, 1H), 6.66 (dd, J = 9.1,

2.8 Hz, 1H), 6.48 (d, J = 2.7 Hz, 1H), 6.40 (s, 1H), 4.00 (s, 3H), 3.97

(s, 3H), 3.47 (q, J = 7.0 Hz, 4H), 1.26 (t, J = 7.1 Hz, 6H). HRMS

(22, APCI⁺) m/z: [M + H]⁺calcd for C₂₃H₂₃N₂O₅407.1601; found

407.1601. HRMS (15, APCI⁺) m/z: [M + H]⁺calcd for C₂₁H₂₁N₂O₃

349.1546; found 349.1547.

̌

́

̌

́

experiments and fruitful discussions and Miroslava Bittova

(Masaryk University) for HRMS measurements.

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https://dx.doi.org/10.1021/acs.joc.9b03217

J. Org. Chem. XXXX, XXX, XXX−XXX

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Structural modifications of nile red carbon monoxide fluorescent probe: Sensing mechanism and applications

DOI: 10.1021/acs.joc.9b03217

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Article abstract of DOI:10.1021/acs.joc.9b03217

Full text of DOI:10.1021/acs.joc.9b03217

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