Fluorescent Probes from Stable Aromatic Nitrile Oxides

Article abstract of DOI:10.1002/ejoc.201501478

Stable aromatic nitrile oxides underwent a 1,3-dipolar cycloaddition reaction with 1-iodo-4-(prop-2-yn-1-yloxy)benzene to afford the expected isoxazoles in very good yields as single regioisomers. The formation of the 5-substituted isoxazoles is in agreement with frontier orbital theory. The reductive cleavage of the N–O bond followed by complexation with BF₃·Et₂O afforded the corresponding boron complexes, which were further derivatized with a triple bond for click chemistry applications. The photochemical behavior and the fluorescence properties of the derivatives were investigated and discussed, taking theoretical calculations into consideration.

Full text of DOI:10.1002/ejoc.201501478

DOI: 10.1002/ejoc.201501478
Full Paper
Fluorescent Probes
Fluorescent Probes from Stable Aromatic Nitrile Oxides
Luigi Ferdinando Minuti,[a] Misal Giuseppe Memeo,[a] Stefano Crespi,[a] and
Paolo Quadrelli*[a]
Abstract: Stable aromatic nitrile oxides underwent a 1,3-di- BF3·Et2O afforded the corresponding boron complexes, which
polar cycloaddition reaction with 1-iodo-4-(prop-2-yn-1-yloxy)-
benzene to afford the expected isoxazoles in very good yields
as single regioisomers. The formation of the 5-substituted isox-
azoles is in agreement with frontier orbital theory. The reductive
cleavage of the N–O bond followed by complexation with
were further derivatized with a triple bond for click chemistry
applications. The photochemical behavior and the fluorescence
properties of the derivatives were investigated and discussed,
taking theoretical calculations into consideration.
Introduction
Nitrile oxides that have sterically encumbered substituents are
known to be remarkably stable, as these bulky groups prevent
the spontaneous dimerization of these compounds into furox-
ans.[1] In particular, stable aromatic nitrile oxides are valuable
1,3-dipoles that can be used for pentatomic heterocyclic syn-
thesis, and despite the inability of these stable nitrile oxides
to dimerize, they display all of the other known properties for
employment in 1,3-dipolar cycloaddition reactions.
The well-established synthetic protocol relies on the acquisi-
tion of commercially available aromatic aldehydes 1 that are
easily converted into the corresponding oximes 2. These latter
compounds can undergo a chlorination reaction in the pres-
ence of chlorine gas or, alternatively, by using N-chlorosuccin-
imide (NCS) in an organic solvent to afford hydroxymoyl chlor-
ides 3, which can either be isolated or immediately converted
in situ into stable nitrile oxides 4 (Scheme 1).[2] Nitrile oxides 4
can be stored at low temperature (approximately 4 °C) for days,
weeks, and even months and are suitable for immediate addi-
tion to an organic solution that contains an alkene or alkyne to
afford the desired isoxazoline or isoxazole 5.[3]
Pentatomic heterocycles 5 are important scaffolds and can
be synthetically elaborated into other compounds by taking
advantage of well-known transformations, such as ring-opening
and subsequent inter- or intramolecular ring-closing reactions
that lead to other heterocyclic systems.[3] However, the ring-
opening reaction alone represents a valuable and general
method for transforming pentatomic heterocycles into aliphatic
compounds to be used for several synthetic applications.
Throughout decades, a number of methods have been pro-
Scheme 1. Synthetic route for the preparation of stable aromatic nitrile oxide
and its 1,3-dipolar cycloaddition reaction with an alkene or alkyne.
posed to exploit the ring-opening transformation of isoxazole 5
into enamino ketone 6 (Scheme 2). For example, the reductive
cleavage of the isoxazole N–O bond has been efficiently per-
formed under reductive conditions by using transition metals
or their complexes in the presence of additives.[4]
The structure and areas of practical applications for enamino
ketones 6 is spread over different fields of study, but their use
as ligands for the coordination of metals takes advantage of
the presence of the amino group and oxygen atom of the
carbonyl group, which have a 1,5-relationship.[5] These features
allow for the easy preparation of highly stable complexes to be
used as catalysts in polymer synthesis.[6]
Recently, the complexation of boron with ligands of type
6 was thoroughly investigated and led to stable ketoiminate
complexes 7, the fluorescence properties of which have at-
tracted a great deal of interest for use as organic diodes and
for other tunable electronic properties.[7] However, one of the
most intriguing applications is their use as chemical probes to
label enzymatic activity, in which the tunable properties of
boron complexes of type 7, controlled by the addition of suit-
able substituents, can be best optimized for studies in bio-
orthogonal chemistry.[8] Herein, we report our approach to new
fluorescent probes that are designed by the synthetic elabora-
[a] Department of Chemistry, University of Pavia,
Viale Taramelli 12, 27100 Pavia, Italy
E-mail: paolo.quadrelli@unipv.it
http://chimica.unipv.eu/site/home/persone/scheda700003454.html
Supporting information and ORCID(s) from the author(s) for this article
are available on the WWW under http://dx.doi.org/10.1002/
ejoc.201501478.
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Scheme 2. Isoxazole reductive ring-opening and boron complexation of corresponding enamino ketones (DMF = N,N-dimethylformamide).
tion of isoxazoles, which are prepared through a 1,3-dipolar methyleneoxy protons of 5a and 5b at δ = 5.36 and 5.28 ppm,
cycloaddition reaction of stable aromatic nitrile oxides.
respectively. Notably, cycloadduct 5a showed a slight fluores-
cence both in solution and in the solid state.
Frontier orbital theory nicely accounts for the origin of the
regioselectivity in these cycloaddition reactions.[1] According to
the principle of maximal overlap, the formation of the preferred
isomers can be predicted by combining the reactant sites that
have the largest coefficients. Almost unidirectional behavior is
observed in the cycloaddition of nitrile oxides to monosubsti-
tuted alkynes, leading to the 5-substituted isoxazoles.[11]
Results
1,3-Dipolar Cycloaddition Reaction and Ligand Synthesis
9-Anthracenenitrile oxide (4a) and 9-phenanthrenenitrile oxide
(4b) were prepared in very good yields (4a: 76 %, 4b: 69 %)
from the corresponding oximes by the standard treatment with
NCS in chloroform solution at 0 °C for 4 h, according to adapted
reported procedures.[1,2,9] The stable solid aromatic nitrile ox-
ides 4a and 4b could be stored for months at low temperature
and were used, without further purification or adaptation to the
experimental procedure, in the 1,3-dipolar cycloaddition with
synthesized 1-iodo-4-(prop-2-yn-1-yloxy)benzene.[10] The cyclo-
addition reactions were conducted in anhydrous dichlorometh-
ane (DCM) as the solvent at room temperature for 48 h
(Scheme 3). The 1,3-dipolar cycloadducts 5a and 5b were iso-
lated by chromatographic purification in 55 and 47 % yield, re-
spectively. Both products were obtained as solids and single
regioisomers.
Typically, selective N–O bond cleavage[7b] of the isoxazole
moiety was performed by using Mo(CO)6 in a mixed solvent of
H2O/CH3CN (70 °C, 6 h) to give enamino ketones 6a and 6b as
dark reddish powders in 75 and 80 % yield, respectively. The
structures of enamino ketones 6a and 6b were determined by
their analytical and spectroscopic data, and the diagnostic sig-
1
nals of the H NMR spectra along with the carbonyl chemical
shifts from the 13C NMR spectra are reported in Table 1. In par-
1
ticular, the signals observed in the H NMR spectra show the
existence of intramolecular hydrogen bonding between the
amino group and the oxygen atom of the carbonyl moiety, as
both functionalities were obtained by the cleavage of the N–O
bond. One of the protons of the NH2 group is observed at δ =
The structures of compounds 5a and 5b were assigned by
their analytical and spectroscopic data. Besides the signals from
the aromatic moieties in the 1H NMR spectra, the diagnostic
signal of the isoxazole ring proton of 5a and 5b was easily
detected at δ = 6.62 and 6.74 ppm, respectively. The inclusion
1
5.57(8) ppm in the H NMR spectrum, and the other is strongly
deshielded at δ = 10.3(4) ppm. In the enamino ketone form of
6, the latter proton participates in a hydrogen bond with the
carbonyl oxygen atom to form a six-membered cyclic array as
of the 4-iodophenoxy moiety into each cycloadduct was also shown in Table 1. The possibility of a tautomeric equilibrium
confirmed by the presence of a singlet that corresponds to the with the iminoenol form, depending upon the solvent, temper-
Scheme 3. 1,3-Dipolar cycloaddition reaction and ligand synthesis.
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ature, and type of substituents, can be excluded in the cases at
hand because of the clear presence of the carbonyl carbon sig-
nals in the 13C NMR spectra.[12]
Table 2. Diagnostic signals of 1H NMR, 13C NMR, and FTIR spectra of boron
complexes 7a and 7b.
Compound
1H NMR[a]
=CH
FTIR[b]
νNH
˜
–CH2–
F2B–NH
7a
7b
4.91
4.88
6.22
6.26
7.80
7.59
3359
3333
Table 1. Diagnostic signals from 1H and 13C NMR spectra of enamino ketones
6a and 6b.[a]
[a] Chemical shifts (δ) are reported in ppm, and CDCl3 was used as the sol-
vent. [b] Nujol mulls of the samples were used to obtain the spectra. Sharp
bands are reported in cm–1
.
When the boron atom is coordinated by the ketoiminate, a
1
single NH proton signal is detected in the H NMR spectrum at
δ > 7 ppm, which is strongly deshielded as a result of the nitro-
gen complexation to the boron atom. In the 13C NMR spectrum,
the signal that corresponds to the free carbonyl group has now
disappeared, presumably because of the longer relaxation time
of this carbon signal of the complex. Significantly, sharp bands
in the FTIR spectra in the range of 3330–3360 cm–1 confirm the
presence of a single NH bond. Finally, the methylene and
methine protons were also deshielded because of the boron
insertion. The methine (=CH) proton was shifted downfield by
approximately 0.5 ppm, whereas the chemical shift of the meth-
ylene proton was only slightly affected by the complex forma-
tion (+0.2 ppm, downfield).
To provide the boron complexes with a linker to which a
possible “chemical reporter” can be anchored through a peri-
cyclic reaction,[8] we derivatized complexes 7a and 7b with a
protected triple bond by using a Sonogashira coupling reaction
(Scheme 4). First, boron complexes 7a and 7b were dissolved
in anhydrous DMF along with an excess amount of triethyl-
amine and ethynyltrimethylsilane in the presence of 10 mol-%
Compound
1H NMR
=CH
13C NMR
C=O
–CH2–
–NH2
6a
6b
4.64
4.61
5.74
5.75
5.58, 10.44
5.57, 10.28
194.2
194.0
[a] Chemical shifts (δ) are reported in ppm, and CDCl3 was used as the NMR
solvent.
Boron Complexation, Sonogashira Coupling, and
Deprotection
4-Amino-1-(4-iodophenoxy)-4-arylbut-3-en-2-ones 6a and 6b
were then used as ligands in a boron complexation[13] process
that employed the addition of 10 equiv. of BF3·Et2O in anhy-
drous DCM solution in the presence of an excess amount of
Et3N (Scheme 4).
Boron complexes 7a and 7b were obtained after chromato- of CuI/Pd(PPh3)4 as the catalyst. The reactions were conducted
graphic purification as yellow solids in very good yields (63 and at room temperature for 1 h under nitrogen. Workup of the
64 %, respectively). The structures were assigned by using their reaction mixture followed by chromatographic purification af-
analytical and spectroscopic data. Both the 1H and 13C NMR forded the expected derivatives 8a and 8b as yellow-orange
spectra gave clear evidence of complex formation, as there oils in 80 and 77 % yield, respectively (Scheme 4).[14] The struc-
were relevant changes to the chemical shifts of the protons tures of 8a and 8b were confirmed by the spectroscopic data.
1
bonded to the atoms involved in the complex. The physical In the H NMR spectrum, the singlet from the TMS group of 8a
data supporting the complex formation are reported in Table 2. and 8b is easily detected at δ = 0.26 and 0.27 ppm, respectively.
Scheme 4. Boron complexation, Sonogashira coupling, and deprotection reactions (TMS = trimethylsilyl, TBAF = tetra-n-butylammonium fluoride).
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The sharp stretching bands of the C≡ C triple bond are visible 1.83 × 104 mol–1. The two aromatic substituents do not signifi-
in the FTIR spectra at approximately ν = 2150 cm–1.
cantly affect the absorption values.
˜
The final deprotection step was achieved by treatment with
The excitation of 9a and 9b at their absorption maxima gen-
TBAF (1 equiv.) in anhydrous tetrahydrofuran (THF) at room erated the fluorescence emission spectra shown in Figure 2.
temperature for 10 min, which gave the desired acetylene de-
rivatives 9a and 9b as yellow solids in 77 and 88 % yield, re-
spectively. In the 1H NMR spectra of 9a and 9b, the signal from
the TMS group disappeared, and the presence of the ≡ C–H pro-
ton was confirmed by the presence of singlets at δ = 3.03 and
3.05 ppm, respectively.[15]
UV/Vis and Fluorescence Studies
To evaluate the potential use of boron complexes 9a and 9b as
chemical probes for biological studies, we recorded the corre-
sponding UV/Vis and fluorescence spectra. In most cases, the
fluorescence was assigned to the π→π* transition localized on
the conjugated chelating backbone, as confirmed by computa-
tional analysis (see below). When the organic core includes
strong donor and acceptor groups, an emissive excited charge-
transfer state can be observed.
Figure 1 shows the UV/Vis spectra of compounds 9a and 9b
in DCM. Anthracene-substituted BF2 complex 9a has an absorp-
tion band at λ = 387 nm (ε = 8.18 × 103 mol–1) along with
vibrational peaks. In contrast, phenanthrene derivative 9b has
the same absorption peak at λ = 324 nm but with a higher ε =
Figure 2. Fluorescence spectra of compounds 9a and 9b in DCM (grey line:
excitation, black line: emission).
The fluorescence maxima appear at λ = 490 nm for 9a and
λ = 422 nm for 9b, indicating a Stokes shift of 100 and 90 nm,
respectively, for the two complexes. Compound 9a has a
fluorescence quantum yield ΦF = 0.60 in DCM, which was deter-
mined by the fluorescent intensity of the reference compound
(see Supporting Information).[13] Analogously, compound 9b
has a ΦF = 0.24 in DCM, which is indicative of modest fluores-
cence properties.
The solvent effects on the absorption and fluorescence prop-
erties were examined for the more promising boron complex
9a (spectra in the Supporting Information), and Table 3 reports
the ΦF values obtained in the selected solvents.
Table 3. Absorption λmax [nm] and fluorescence quantum yield (ΦF) values
for boron complex 9a in the listed solvents.[a]
Solvent
Dielectric
constant
Viscosity[16]
at 20 °C [cP]
λmax
[nm]
λem
[nm]
ΦF
DCM
MeOH
8.9
33
39
55
0.44
0.55
23.5
3.30
385
384
387
388
490
445
452
453
0.60
0.08
0.07
0.42
Ethylene glycol
DMSO[b]/H2O
Figure 1. UV/Vis spectra of compounds 9a and 9b in DCM.
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[a] Concentration of 2 × 10–5
M in all solvents. [b] DMSO = dimethyl sulfoxide.
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The absorption λmax values of 9a were only slightly affected glet at δ = 5.63 ppm from the benzylic methylene protons and
by solvent polarity, which suggests that the dipole moments of all of the other signals are in agreement with the assigned
the molecule in the ground and excited states were almost the structure. The success of this experiment reveals the potential
same.[18] On the other hand, the maximum fluorescence λem use of these compounds in click reactions for the purpose of
values are blueshifted approximately 40 nm in polar solvents biological monitoring in the presence of azidopeptides or azide
relative to the value in DCM. This reduced the Stokes shift by derivatives of chemical reporters in protein profiling experi-
approximately 64 nm.
ments.[8]
Significantly, the ΦF values dramatically changed with varia-
tions in the solvent polarity as well as viscosity. Remarkable
drops in the quantum yields are observed in MeOH (0.08) and
ethylene glycol (0.07) relative to that in DCM. However, in the
DMSO/H2O (1:1) mixture, there was just a modest decrease in
the ΦF value (0.42), which indicates potential for using this
compound in ambient biological systems.
Finally, one of the most striking properties of these com-
plexes is its retention of fluorescence in the solid state. In fact,
complex 9a was also fluorescent in the solid state (Figure 3),
which points to applications in electronics rather than biology.
Discussion
The experiments in this study were designed to promote the
use of stable aromatic nitrile oxides in the simple and reliable
synthesis of boron fluorescent complexes for potential use in
live-cell imaging.
In the literature, reports regarding imaging probe develop-
ment account for a variety of fluorescent molecules used as
sensors and probes, which were initially prepared by target-
oriented syntheses.[17] Diversity-oriented approaches are de-
signed to overcome the limitations imposed by a systematic
synthesis. Of the structures and optical properties that have
been reviewed, isoquinoline and xanthone are typically in the
blue range, and chalcone is in the green. Rosamine dyes are
usually in yellow to orange range, and boron-dipyrromethene
(BODIPY) dyes span a broad range from green to red, depend-
ing on the electronic structure of the conjugated aromatic
group.[18]
Among heterocyclic ligand-based boron complexes, a large
number have emission wavelengths that span 400–500 nm, and
compounds 9a and 9b can be considered part of this group,
which often contains pyridine, quinolone, and carbazole rings,
just to cite those most frequently found.[19] Recently, anthra-
cene-substituted BODIPYs were also proposed as optically and
thermally switchable electronic compounds, which have the an-
thracene moiety bonded to the BODIPY core at the 2-position
of the aromatic system instead of the 9-position, as found in
compound 9a.[20]
Figure 3. Solid state fluorescence of compound 9a.
To gain further insight into the photochemical behavior of
boron complexes 9a and 9b and provide a reasonable rationale
for their different fluorescence properties, we performed DFT
theoretical calculations at the B3LYP/6-31G(d) level[21] to opti-
mize the structures of the fluorescent molecules in both the
gas phase and with DCM as the solvent.[22] No symmetry con-
Click Chemistry Test
A click chemistry test was performed with boron complex 9a
and benzyl azide in the presence of catalytic copper sulfate
and sodium ascorbate in DCM at room temperature for 2 h
(Scheme 5).
The expected cycloadduct was isolated from the reaction straint was applied to the investigated structures, and fre-
mixture as a yellow fluorescent solid in 60 % yield. The structure quency calculations were performed in vacuo to be sure that
was assigned by using the corresponding analytical and spec- the minima had no imaginary frequencies. Figure 4 shows the
troscopic data. In the 1H NMR spectrum, new signals corre- electronic absorption spectra of compounds 9a and 9b,
sponding to the 1-benzyl-1,2,3-triazole ring were present. A sin- which were calculated as vertical excitations from the minima
Scheme 5. 1,3-Dipolar cycloaddition reaction between compound 9a and benzyl azide.
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of the ground-state structure by using the TDDFT//B3LYP/6- compounds 9a and 9b (figures, calculated structures, and en-
311G+(d,p) approach.
ergy values are reported in the Supporting Information) involve
a charge-transfer from the aromatic moiety to the boron six-
membered ring. Interestingly, the LUMO and LUMO+1 orbitals
for compound 9a are nearly degenerated, and the LUMO+1
orbital presents a more marked π* character, which belongs to
the anthracene moiety.
The calculated spectra nicely resemble the experimental ab-
sorption spectra of Figure 1 and have transition frequencies at
λ = 394 and 370 nm, respectively, for 9a and 9b. The two lower
transitions for compound 9a are quanto-mechanically allowed
(fS0→S1 = 0.0123 and fS0→S2 = 0.1203), as is the S0→S1 transition
(f = 0.0654) for compound 9b. This means that both the afore-
mentioned compounds are to be considered fluorescent, which
is in agreement with the experimental results. A more detailed
description of the states involved in the transitions is provided
for compound 9a and 9b in the Supporting Information.
However, the differences in the experimental ΦF values of
the two products can be explained by the existence of nonradi-
ative deactivation pathways that occur through the rotation of
the side groups.[19] For this reason, we have also calculated the
rotational barriers of the anthracene and phenanthrene moie-
ties. A relaxed scan [B3LYP/6-31G(d)] of the dihedral angle C-1–
C-2–C-3–C-4 was conducted to access the rotational barriers
of these aromatic groups around the C-2–C-3 bond (Figure 5).
Compound 9a has the highest rotational barriers at 10°
(29.6 kcal mol–1) and 210° (28.8 kcal mol–1), which confirms the
known tendency of the anthracene group to be out of the
plane in linked conjugated structures.
Figure 4. Calculated electronic absorption spectra of compounds 9a (top) and
9b (bottom).
The solvent effects were evaluated by using the conductor-
like approach within the framework of the polarizable contin-
uum models (CPCM, DCM bulk).[23] The solvent cavity was calcu-
lated by using the united atom topological model applied on
radii optimized for the HF/6-31G(d) level of theory (RADII =
UAHF option).
Compound 9b shows its highest rotational barriers at 11°
(23.4 kcal/mol) and 206° (23.6 kcal/mol), both characterized by
lower energies. Hence, it is less difficult for the phenanthrene
group to rotate around the C–C bond. The higher conforma-
The time-dependent density functional theory (TDDFT) anal- tional mobility of the phenanthrene group may, to some extent,
ysis in DCM clearly shows that the HOMO–LUMO transitions of explain the lower fluorescence of product 9b.
Figure 5. Rotational barriers of compounds 9a (top) and 9b (bottom). The dihedral angles C-1–C-2–C-3–C-4 for which the relaxed scans were computed (see
black arrow) are highlighted.
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NMR (75 MHz, CDCl3): δ = 61.6, 84.3, 107.0, 117.2, 121.2, 125.3,
125.4, 126.5, 128.5, 129.0, 130.5, 131.0, 138.4, 157.6, 160.7,
167.7 ppm. C24H16INO2 (477.29): calcd. C 60.39, H 3.38, N 2.93; found
C 60.35, H 3.40, N 2.95.
Conclusions
It has been told that multidisciplinary research on new lumines-
cent dyes is mainly propelled by electronic and biomedical
science.[19] Although the chemistry of isoxazoles in the field of
imaging probes is well known, deeply rooted in the topic of
1,3-dipolar cycloadditions, and has recently attracted much at-
tention, the number of related papers and patents remains
quite low (few sets of ten).[24] Our work has demonstrated the
possibility to expand the application of 1,3-dipolar cycloaddi-
tion reactions of stable aromatic nitrile oxides as a fast ap-
proach to new fluorescent structures. The synthesis is clearly
reliable and provides easily derivatized boron complexes, as
demonstrated by the further cycloaddition reaction using a
click chemistry approach, typically found in biochemistry. Im-
provements on the fluorescence properties of enamino ketone
based boron complexes are being actively pursued, and prelim-
inary tests with lysate to detect the presence of system interfer-
ences with bioorthogonal ligation have been planned.
Cycloadduct 5b: White solid (1.57 g, 47 %); m.p. 127–130 °C (cyclo-
hexane/ethyl acetate). IR: νC=N = 1568 cm–1 1H NMR (300 MHz,
.
˜
CDCl3): δ = 5.28 (s, 2 H, CH2), 6.74 (s, 1 H, =CHisox.), 6.83 and 7.63
(AA′BB′ syst., 2 H + 2 H, 4-iodophenyl), 7.66 (m, 2 H, Ar), 7.75 (t, J =
7 Hz, 2 H, Ar), 7.96 (d, J = 7 Hz, 1 H, Ar), 8.01 (s, 1 H, Ar), 8.36 (d,
J = 7 Hz, 1 H, Ar), 8.74 (d, J = 7 Hz, 1 H, Ar), 8.80 (d, J = 7 Hz, 1 H,
Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = 61.4, 84.2, 105.1, 117.1,
122.5, 123.0, 125.2, 126.1, 126.9, 127.0, 127.1, 127.8, 129.1, 129.3,
129.4, 130.6, 130.7, 130.8, 138.4, 157.5, 162.3, 167.2 ppm. C24H16I-
NO2 (477.29): calcd. C 60.39, H 3.38, N 2.93; found C 60.41, H 3.37,
N 2.96.
4-Amino-4-(anthracen-9-yl)-1-(4-iodophenoxy)but-3-en-2-one
(6a) and 4-Amino-1-(4-iodophenoxy)-4-(phenanthren-9-yl)but-
3-en-2-one (6b): Cycloadduct 5a or 5b (0.74 g, 1.4 mmol) was dis-
solved in a mixture of acetonitrile/water (9:1, 100 mL). Under nitro-
gen, the solution was stirred for 15 min, and then Mo(CO)6 (0.37 g,
1.4 mmol) was added. The resulting mixture was heated at 70 °C
for 4 h. DCM was added, and the mixture was washed with brine
(3×). The combined organic phases were dried with anhydrous
Na2SO4, and the solvent was evaporated. The crude residue was
purified by column chromatography to give enamino ketone 6a or
6b.
Experimental Section
General Methods: Elemental analyses were performed with an ele-
mental analyzer that was available in the department. The H and
1
13C NMR spectroscopic data were recorded with a Bruker AVANCE
300 MHz spectrometer (solvents are specified). Chemical shifts (δ)
are reported in ppm by using tetramethylsilane as the internal
standard, and coupling constants (J) are reported in Hertz. The fol-
lowing abbreviations are used to describe the multiplicities of the
NMR signals: br. (broad), s (singlet), d (doublet), t (triplet), and m
(multiplet). IR spectra (Nujol mulls for solid compounds) were re-
corded on a Perkin–Elmer RX-1 spectrophotometer, which was avail-
Enamino Ketone 6a: Reddish solid (0.50 g, 75 %); m.p. 156–160 °C
(cyclohexane/ethyl acetate). IR: νNH2 = 3457 (and 3261) cm–1 1H
.
˜
NMR (300 MHz, CDCl3): δ = 4.64 (s, 2 H, CH2), 5.58 (br. s, 1 H, HN-
H), 5.74 (s, 1 H, C=CH), 6.70 and 7.57 (AA′BB′ syst., 2 H + 2 H, 4-
iodophenyl), 7.52 (m, 4 H, Ar), 8.05 (m, 4 H, Ar), 8.53 (s, 1 H, Ar),
10.44 (br. s, 1 H, H-NH) ppm. 13C NMR (75 MHz, CDCl3): δ = 71.7,
83.2, 95.6, 117.0, 125.1, 125.5, 126.7, 128.3, 128.5, 130.9, 138.3, 162.0,
194.2 ppm. C24H18INO2 (479.31): calcd. C 60.14, H 3.79, N 2.92; found
C 60.15, H 3.71, N 2.95.
able in the department, and the wavenumbers (ν) of the bands are
˜
given in cm–1. UV/Vis spectroscopy was carried out on a Jasco V-
550 spectrophotometer, and fluorescence spectra were recorded on
a Perkin–Elmer LS 55 luminescence spectrometer. Column chroma-
Enamino Ketone 6b: Reddish solid (0.54 g, 80 %); m.p. 146–148 °C
(cyclohexane/ethyl acetate). IR: νNH2 = 3384 (and 3260) cm–1 1H
.
˜
tography and TLC were carried out on silica gel H60 and GF254
,
NMR (300 MHz, CDCl3): δ = 4.61 (s, 2 H, CH2), 5.57 (br. s, 1 H, HN-
H), 5.75 (s, 1 H, C=CH), 6.72 and 7.56 (AA′BB′ syst., 2 H + 2 H, 4-
iodophenyl), 7.65 (m, 5 H, Ar), 7.91 (d, J = 7 Hz, 1 H, Ar), 8.11 (d, J =
7 Hz, 1 H, Ar), 8.71 (m, 2 H, Ar), 10.28 (br. s, 1 H, H-NH) ppm. 13C
NMR (75 MHz, CDCl3): δ = 61.4, 83.2, 105.1, 117.0, 122.5, 123.0,
125.7, 126.1, 126.8, 127.0, 127.1, 127.7, 128.5, 129.0, 129.3, 130.5,
130.6, 133.6, 138.2, 158.1, 163.4, 194.0 ppm. C24H18INO2 (479.31):
calcd. C 60.14, H 3.79, N 2.92; found C 60.16, H 3.73, N 2.90.
respectively, by using a gradient elution from cyclohexane/ethyl ac-
etate (9:1) to pure ethyl acetate, unless otherwise stated. A Biotage
apparatus equipped with KP-SIL columns was used for medium
pressure liquid chromatography (MPLC). All reagents and solvents
were purchased from Sigma–Aldrich and used without any further
purification. The syntheses of nitrile oxides 4a and 4b are described
in the Supporting Information.
3-(Anthracen-9-yl)-5-[(4-iodophenoxy)methyl]isoxazole
(5a)
Synthesis of Boron Complexes 7a and 7b: Enamino ketone 6a or
6b (1.65 g, 3.6 mmol) was dissolved in anhydrous DCM (50 mL)
along with distilled triethylamine (5 mL, 36 mmol). The solution was
kept under an inert atmosphere and stirred for 10 min. BF3·Et2O
(4.5 mL, 36 mmol) was added, and the mixture was kept at room
temperature for 2 h. DCM was added, and the mixture was then
washed with brine (3×). The combined organic phases were dried
with anhydrous Na2SO4, and the solvent was evaporated. The result-
ing dark oily residue was purified by column chromatography to
give boron complexes 7a or 7b.
and 5-[(4-Iodophenoxy)methyl]-3-(phenanthren-9-yl)isoxazole
(5b): To 1-iodo-4-(prop-2-yn-1-yloxy)benzene (1.8 g, 7 mmol) dis-
solved in anhydrous DCM (100 mL) was added a solution of nitrile
oxide 4a or 4b (1.64 g, 7.4 mmol) in anhydrous DCM (50 mL) drop-
wise, as the reaction was stirred. The resulting mixture was stirred
and kept in the dark at room temperature for 48 h. DCM was then
added, and the mixture was washed with brine (3×). The combined
organic phases were then dried with anhydrous Na2SO4, and the
solvent was evaporated. The crude residue was purified by column
chromatography to give cycloadduct 5a or 5b.
4-(Anthracen-9-yl)-2,2-difluoro-6-[(4-iodophenoxy)methyl]-2,3-
Cycloadduct 5a: White solid (1.84 g, 55 %); m.p. 131–134 °C (cyclo- dihydro-1,3,2-oxazaborinin-1-ium-2-uide (7a): Yellow solid
hexane/ethyl acetate). IR: νC=N = 1593 cm–1
.
1H NMR (300 MHz,
(1.20 g, 63 %); m.p. 157–161 °C (cyclohexane/ethyl acetate). IR:
˜
1
CDCl3): δ = 5.36 (s, 2 H, CH2), 6.62 (s, 1 H, =CHisox.), 6.87 and 7.66
(AA′BB′ syst., 2 H + 2 H, 4-iodophenyl), 7.53 (m, 4 H, Ar), 7.83 (d, J =
νNH = 3359 cm–1. H NMR (300 MHz, CDCl3): δ = 4.91 (s, 2 H, CH2),
˜
6.22 (s, 1 H, C=CH), 6.70 and 7.58 (AA′BB′ syst., 2 H + 2 H, 4-iodophe-
7 Hz, 2 H, Ar), 8.08 (d, J = 7 Hz, 2 H, Ar), 8.61 (s, 1 H, Ar) ppm. 13C nyl), 7.55 (m, 4 H, Ar), 7.80 (s, 1 H, NH), 7.83 (m, 2 H, Ar), 8.08 (s, 2
Eur. J. Org. Chem. 2016, 821–829
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H, Ar), 8.63 (s, 1 H, Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = 67.5,
1 H, ≡ CH), 4.93 (s, 2 H, CH2), 6.23 (s, 1 H, C=CH), 6.83 and 7.42 (AA′
84.2, 97.8, 116.9, 123.8, 125.5, 125.9, 127.6, 127.8, 127.9, 128.8, 130.4, BB′ syst., 2 H + 2 H, Ar), 7.52 (m, 4 H, Ar), 7.62 (br. s, 1 H, NH), 7.83
130.7, 138.4, 157.1, 173.7, 177.1 ppm. C24H17BF2INO2 (527.11): calcd. (m, 2 H, Ar), 8.07 (m, 2 H, Ar), 8.60 (s, 1 H, Ar) ppm. 13C NMR (75 MHz,
C 54.69, H 3.25, N 2.66; found C 54.65, H 3.21, N 2.65.
CDCl3): δ = 55.7, 67.4, 83.0, 114.5, 117.2, 123.8, 125.9, 127.8, 127.9,
128.3, 128.8, 130.4, 130.7, 133.7, 138.2, 157.5, 173.7, 177.2 ppm.
2,2-Difluoro-6-[(4-iodophenoxy)methyl]-4-(phenanthren-9-yl)-
2,3-dihydro-1,3,2-oxazaborinin-1-ium-2-uide (7b): Yellow solid
C26H18BF2NO2 (425.23): calcd. C 73.44, H 4.27, N 3.29; found C 73.45,
H 4.22, N 3.25.
(1.21 g, 64 %); m.p. 170–175 °C (cyclohexane/ethyl acetate). IR:
1
νNH = 3333 cm–1. H NMR (300 MHz, CDCl3): δ = 4.88 (s, 2 H, CH2),
6-[(4-Ethynylphenoxy)methyl]-2,2-difluoro-4-(phenanthren-9-
˜
6.26 (s, 1 H, C=CH), 6.75 and 7.61 (AA′BB′ syst., 2 H + 2 H, 4-iodophe- yl)-2,3-dihydro-1,3,2-oxazaborinin-1-ium-2-uide (9b): Yellow
nyl), 7.59 (s, 1 H, NH), 7.77 (m, 5 H, Ar), 7.88 (d, J = 7 Hz, 1 H, Ar), solid (0.45 g, 88 %); m.p. 150–152 °C (cyclohexane/ethyl acetate). IR:
1
7.98 (d, J = 7 Hz, 1 H, Ar), 8.72 (d, J = 7 Hz, 1 H, Ar), 8.79 (d, J =
ν = 3369 (NH), 3304 (CH) cm–1. H NMR (300 MHz, CDCl3): δ = 3.05
˜
7 Hz, 1 H, Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = 67.4, 84.2, 96.2,
(s, 1 H, ≡ CH), 4.92 (s, 2 H, CH2), 6.27 (s, 1 H, C=CH), 6.88 and 7.42
116.9, 122.7, 123.5, 124.9, 127.0, 127.6, 127.7, 127.9, 128.3, 129.1, (AA′BB′ syst., 2 H + 2 H, Ar), 7.75 (m, 4 H, Ar), 7.72 (m, 1 H, Ar), 7.88
129.7, 130.7, 131.4, 138.5, 157.2, 172.9, 176.4 ppm. C24H17BF2INO2
(527.11): calcd. C 54.69, H 3.25, N 2.66; found C 54.70, H 3.26, N
2.64.
(br. s, 1 H, NH), 7.90 (d, J = 7 Hz, 1 H, Ar), 8.73 (d, J = 7 Hz, 1 H, Ar),
8.79 (d, J = 7 Hz, 1 H, Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = 67.4,
83.0, 96.1, 114.6, 122.7, 123.5, 124.9, 127.6, 127.7, 127.9, 128.2, 129.1,
129.6, 129.7, 133.7, 157.6, 172.9, 176.5 ppm. C26H18BF2NO2 (425.23):
calcd. C 73.44, H 4.27, N 3.29; found C 73.43, H 4.29, N 3.27.
Sonogashira Coupling of Boron Complexes 7a and 7b: Boron
complex 7a or 7b (1.6 g, 3 mmol) was dissolved in anhydrous DMF
(6 mL) along with distilled triethylamine (0.7 mL, 5 mmol) and eth-
ynyltrimethylsilane (0.56 mL, 4 mmol) in the presence of CuCl
(0.16 g, 10 mol-%) and Pd(PPh)4 (0.16 g, 10 mol-%). The mixture was
stirred for 2 h under an inert atmosphere. DCM was added, and the
mixture was washed with a saturated solution of NH4Cl (3 × 50 mL)
and then brine (3×). The combined organic phases were dried with
anhydrous Na2SO4, and the solvent was evaporated. The resulting
black oily residue was purified by column chromatography to give
the functionalized boron complex 8a or 8b.
Cycloaddition of Benzyl Azide to Compounds 9a: To boron com-
plex 9a (0.1 g, 0.2 mmol) dissolved in anhydrous DCM (40 mL) was
added benzyl azide (0.4 mL, 0.2 mmol), as the mixture was stirred
under an inert atmosphere. A catalytic amount of CuSO4·10H2O was
added to the solution along with the same quantity of sodium
ascorbate. The mixture was then allowed to react at room tempera-
ture for 2 h. DCM was then added, and the resulting mixture was
washed with brine (3 × 50 mL). The organic phase was dried with
anhydrous Na2SO4, and the solvent was evaporated. The resulting
yellow oily residue was purified by column chromatography to give
cycloadduct 10.
4-(Anthracen-9-yl)-2,2-difluoro-6-({4-[(trimethylsilyl)ethynyl]-
phenoxy}methyl)-2,3-dihydro-1,3,2-oxazaborinin-1-ium-2-uide
= 3296 cm–1 1H NMR
.
4-(Anthracen-9-yl)-6-{[4-(1-benzyl-1H-1,2,3-triazol-4-yl)phen-
oxy]methyl}-2,2-difluoro-2,3-dihydro-1,3,2-oxazaborinin-1-
ium-2-uide (10): Yellow solid (0.07 g, 60 %); m.p. 175–178 °C
(8a): Yellow oil (1.19 g, 80 %). IR: ν
˜
NH
(300 MHz, CDCl3): δ = 0.26 (s, 9 H, TMS), 4.94 (s, 2 H, CH2), 6.24 (s,
1 H, C=CH), 6.83 and 7.41 (AA′BB′ syst., 2 H + 2 H, Ar), 7.56 (m, 4 H,
Ar), 7.72 (br. s, 1 H, NH), 7.82 (m, 2 H, Ar), 8.09 (m, 2 H, Ar), 8.62 (s,
1 H, Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = –0.10, 67.4, 97.8, 114.4,
123.8, 125.9, 127.6, 127.8, 127.9, 128.8, 130.3, 130.7, 133.5, 157.2,
173.7, 177.2 ppm. C29H26BF2NO2Si (497.41): calcd. C 70.02, H 5.27,
N 2.82; found C 70.05, H 5.22, N 2.85.
(cyclohexane/ethyl acetate). IR: νC=N
= .
1633 cm–1 1H NMR
˜
(300 MHz, [D6]DMSO): δ = 5.05 (s, 2 H, CH2), 5.63 (s, 2 H, CH2), 6.08
(s, 1 H, C=CH), 7.08 and 7.76 (AA′BB′ syst., 2 H + 2 H, Ar), 7.36 (m,
5 H, Ar), 7.60 (m, 4 H, Ar), 7.72 (m, 2 H, Ar), 8.21 (m, 2 H, Ar), 8.54
(s, 1 H, Ar), 8.85 (s, 1 H, NH), 11.15 (s, 1 H, NH) ppm. 13C NMR
(75 MHz, [D6]DMSO): δ = 53.0, 67.0, 97.6, 115.2, 120.8, 123.8, 124.2,
125.9, 126.5, 127.2, 127.7, 127.9, 128.1, 128.2, 128.8, 129.5, 130.4,
136.1, 146.4, 157.0, 172.0, 175.4 ppm. C33H25BF2N4O2 (558.39): calcd.
C 70.98, H 4.51, N 10.03; found C 70.95, H 4.52, N 10.05.
2,2-Difluoro-4-(phenanthren-9-yl)-6-({4-[(trimethylsilyl)-
ethynyl]ohenoxy}methyl)-2,3-dihydro-1,3,2-oxazaborinin-1-
1
ium-2-uide (8b): Yellow oil (1.15 g, 77 %). IR: νNH = 3301 cm–1. H
˜
NMR (300 MHz, CDCl3): δ = 0.27 (s, 9 H, TMS), 4.87 (s, 2 H, CH2),
6.23 (s, 1 H, C=CH), 6.87 and 7.79 (AA′BB′ syst., 2 H + 2 H, Ar), 7.56
(m, 4 H, Ar), 7.6–7.9 (m, 7 H, Ar), 7.86 (br. s, 1 H, NH), 8.68 (m, 1 H,
Ar), 8.74 (m, 1 H, Ar) ppm. 13C NMR (75 MHz, CDCl3): δ = –0.07,
67.3, 96.2, 114.5, 122.6, 123.4, 125.0, 127.0, 127.1, 127.6, 127.7, 128.2,
128.3, 128.5, 128.6, 130.6, 131.3, 133.6, 157.3, 172.8, 176.2 ppm.
C29H26BF2NO2Si (497.41): calcd. C 70.02, H 5.27, N 2.82; found C
70.00, H 5.26, N 2.84.
Acknowledgments
Financial support was provided by the University of Pavia, the
Itakian Ministero dell'Università e della Ricerca (MIUR) (PRIN
2011, CUP: F11J12000210001), and COST Action CM1004. A
warm thank you is also given to Steroid S. p. A. for the generous
research grant.
Deprotection of the Triple Bond – Preparation of Compounds
9a and 9b: To boron complex 8a or 8b (0.6 g, 1.2 mmol) dissolved
in anhydrous diethyl ether (30 mL) was added TBAF (1
M in THF,
Keywords: Fluorescent probes · Boron · Nitrogen
heterocycles · Cycloaddition · UV/Vis spectroscopy
1.2 mL, 1.2 mmol), and the mixture was stirred under an inert at-
mosphere for 10 min. DCM was then added, and the resulting mix-
ture was washed with water. The organic phase was dried with
anhydrous Na2SO4, and the solvent was evaporated. The resulting
yellow oily residue was purified by column chromatography to give
the deprotected boron complex 9a or 9b.
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Received: November 23, 2015
Published Online: December 23, 2015
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim