Synthesis and conformational dynamics of the reported structure of xylopyridine A

Article abstract of DOI:10.1021/ja404737q

Natural products have served as a rich source for the discovery of new nucleic acid targeting molecules for more than half a century. However, our ability to design molecules that bind nucleic acid motifs in a sequence- and/or structure-selective manner i

Full text of DOI:10.1021/ja404737q

Article
pubs.acs.org/JACS
Synthesis and Conformational Dynamics of the Reported Structure
of Xylopyridine A
Robert-Andre F. Rarig, Mai N. Tran, and David M. Chenoweth*
́
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States
*
S Supporting Information
ABSTRACT: Natural products have served as a rich source for the
discovery of new nucleic acid targeting molecules for more than half
a century. However, our ability to design molecules that bind
nucleic acid motifs in a sequence- and/or structure-selective manner
is still in its infancy. Xylopyridine A, a naturally occurring molecule
of unprecedented architecture, has been found to bind DNA by a
unique mode of intercalation. Here we show that the structure
proposed for xylopyridine A is not consistent with the character-
ization in the original isolation report and does not bind B-form
DNA. Instead, we report that the originally proposed structure for
xylopyridine A represents a new class of conformationally dynamic structure-selective quadruplex nucleic acid binder. The unique
molecular conformation locks out nonspecific intercalative binding modes and provides a starting point for the design of a new
class of structure-specific nucleic acid binder.
INTRODUCTION
■
For more than 50 years, natural products have provided a rich
source for the discovery of nucleic acid binding molecules,
some of which have made far reaching impacts in human
medicine ranging from cancer chemotherapy to infectious
1
−10
disease.
Despite half a century of research, our ability to
design small molecules to target nucleic acids in a structure-
and sequence-specific manner, beyond the two primary modes
of recognition (i.e., intercalation and groove binding) is still in
1
−14
its infancy.
Targeting DNA and RNA motifs in a structure-
and sequence-selective manner allows for small molecule
control over cellular events and could provide fundamental
insight into biological processes, paving the way for new
therapeutic strategies. The development of structure- and
sequence-specific small molecule nucleic acid binders repre-
sents an important challenge at the frontier of chemical biology,
where natural products have historically served as inspira-
Figure 1. (a) Chemical structure reported for xylopyridine A (E-1).
b) Relative ground-state free energies calculated using DFT B3LYP
and M06 (basis set = 6-311+G(2d,p)) for the anti-folded and twisted
conformers. (c) X-ray structure of diazaxanthylidene E-1 determined
in this report.
(
1
−14
tion.
Xylopyridine A, a dimeric azaxanthone isolated in 2009 from
the mangrove fungus Xylaria sp. (#2508), was reported as
compound E-1 (Figure 1a) and was found to have potent B-
15−19
15
form DNA binding ability.
Xanthones and azaxanthones
twisted on the basis of AM1 calculations. Xylopyridine A was
also reported to be emissive, a property used to establish DNA
are prevalent in natural products, have been shown to bind
DNA, and have a rich history in medicinal chemistry with
applications in a wide range of therapeutic areas, such as cancer,
1
5
binding using direct fluorimetric titration. The combination
of unprecedented natural product architecture, nucleic acid
binding ability, and unique conformational properties makes
xylopyridine A an important target for synthesis and structural
investigations. Motivated by these unique aspects and the
possibility of accessing xylopyridine A derivatives, we initiated
20−22
cardiovascular disease, and infectious diseases.
Despite this
prevalence, the dixanthylidene (reductively dimerized xan-
thone) architectures have remained elusive in nature until
1
5−19
recently.
The reported diazaxanthylidene structure for
xylopyridine A was primarily established using NMR spectros-
copy, and its reported optical rotation implied a configuration-
ally stable helically chiral conformation, originally assigned as E-
Received: May 11, 2013
©
XXXX American Chemical Society
A
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Article
synthetic studies to shed light on this novel class of nucleic acid
binder.
hexachloroethane in THF to afford 3-chloro-4-cyanopyridine.
The chloride was displaced using sodium phenoxide in DMF to
provide biaryl ether 3, which was heated to 210 °C in
polyphosphoric acid to provide azaxanthone 4 in 56% yield
over three steps. Neither McMurry conditions nor Woollins
reagent effected efficient homodimerization of 4. Lawesson’s
reagent was employed to convert 4 to its corresponding thione
Here, we report the synthesis of the proposed structure for
xylopyridine A and show that E-1 is in fact a conformationally
dynamic structure with an isomerization barrier accessible at
room temperature. X-ray crystallography revealed an anti-
folded conformation in the solid state (Figure 1c), while DFT
calculations and variable temperature NMR studies support a
dynamic equilibrating mixture of olefin isomers in solution.
Experimental and computational UV−vis spectroscopy data
support the minimum energy conformer as anti-folded in the
solid state and in solution. We show that the structure
proposed in the original report of xylopyridine A is not
consistent with our analytical data and its true identity remains
unresolved. Furthermore, we show that the structure proposed
for xylopyridine A is not capable of B-DNA binding. Despite
the inability of E-1 to bind B-DNA, we find that a simple
modification produces a new class of structure-specific G-
quadruplex-binding scaffold that exhibits near perfect specificity
over B-form DNA binding.
24
5 in 97% yield. Homodimerization of thione 5 was effected by
25
reductive coupling using copper powder in refluxing toluene
to afford 1 as a 1.1:1.0 mixture of E and Z isomers in 75% yield
(Scheme 1). The yield over five steps for E-1/Z-1 was 41%.
The isomeric mixture of E-1/Z-1 was inseparable by normal
phase, reverse phase, and supercritical fluid chromatography.
The mixture was characterized by mass spectrometry (MS),
differential scanning calorimetry (DSC), and NMR, UV−vis,
fluorescence, and IR spectroscopy. To our surprise, only our
MS data were consistent with the characterization data reported
1
5
for xylopyridine A. The melting point reported for
15
xylopyridine A was 200−202 °C; however, using DSC, we
found the melting point of crystalline E-1 to be 327 °C, with
decomposition observed above 330 °C (see Supporting
Information). A direct NMR comparison was not viable due
RESULTS AND DISCUSSION
■
Prior to initiating synthetic investigations, we used DFT
calculations to compare the ground-state structures of the most
stable conformers of xylopyridine A (E-1) and its Z isomer (Z-
to the insolubility of E-1 in acetone-d
verify the identity of our dimerization product, single crystals
were obtained by slow evaporation from CHCl /CH CN. X-
6
at room temperature. To
3
3
2
3
1
). The ground-state structures are shown in Figure 1b, with
relative energies using two well-established DFT functionals
see Figure 1b and Supporting Information Table S1). Our
ray data confirmed the structure as E-1 and revealed the anti-
folded conformation E-1-af (Figure 1c), which is consistent
with the relative ground-state energies from DFT calculations
(
1
calculations predict that the E-anti-folded conformation is the
most stable (0.0 kcal/mol), with the Z-anti-folded conforma-
tion very close in energy (0.5−0.6 kcal/mol). Interestingly, the
twisted conformation in both the E and Z isomers is higher in
energy by 4.8−6.0 kcal/mol relative to the anti-folded
conformers. Based on these calculations, indicating the
possibility of an E/Z mixture in an anti-folded conformation,
we initiated our synthetic studies.
(Figure 1b). Dissolving crystals of E-1 at −10 °C provided a H
NMR spectrum identical to that of the isomeric mixture,
indicating isomer equilibration on an experimentally relevant
time scale.
1
Variable-temperature H NMR spectroscopy was used to
investigate the dynamic relationship between Z-1 and E-1
26
(Figure 2). Cooling the sample to −30 °C resulted in
sharpening of unresolved doublets at δ 8.26, indicating
negligible exchange on the NMR time scale. These doublets
are assigned to the pyridine ring and correspond to hydrogens
at the 7 and 7′ positions (Figure 1a). Incremental heating up to
50 °C led to signal broadening indicating slow exchange on the
Given the symmetric nature of xylopyridine A, we envisioned
a rapid synthesis of E-1/Z-1 via reductive homodimerization of
3
-azaxanthone 4 (Scheme 1). Our synthesis began with
chlorination of commercially available 4-cyanopyridine 2 via
lithiation with LiTMP and subsequent treatment with
Scheme 1. Synthesis of the Reported Structure for
Xylopyridine A
Figure 2. Dynamic NMR showing the temperature-dependent
exchange process between isomers Z-1 and E-1. Variable-temperature
NMR experiments conducted in DMF-d7.
B
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Journal of the American Chemical Society
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Figure 3. (a) Absorption spectrum overlaid with results from excited state calculations. All excited state calculations were performed with the 6-
11+G(2d,p) basis set. The height of each marker corresponds to the oscillator strength at each level of theory. Results for both E-1 and Z-1, at each
level of theory, were less than 0.29 eV (2.4 nm) apart and appear as coincidental lines on the plot above. Absorption data were collected at 23 °C
3
−
1
−1
using CHCl as the solvent, Abs λ = 375 nm (ε = 16 659 ± 86 M cm ). (b) Emission and excitation spectra are shown for E-1/Z-1 in CHCl at
3
max
3
2
3 °C (Em λmax = 435 nm, Ex λmax = 378 nm), and the quantum yield in CHCl was found to be Φ = 0.088 ± 0.002.
3
3
8
NMR time scale. Further heating to 100 °C provided a
significantly sharpened spectrum due to fast exchange (Figure
range-separated functionals such as ωB97X-D were chosen.
Results from theoretical calculations predict the absorption
spectrum for the anti-folded isomers to be coincidental and
centered (at 377 nm) between 4.2 and 2.7 eV (292 and 462
nm). The twisted conformation is predicted to be red-shifted
compared to the anti-folded conformation by a mean value of
nearly 1.6 eV (295 nm) to a range between 555 and 758 nm.
Neither the UV absorbance of our synthesized compound (375
nm) nor the λmax of the previously reported species (398 nm)
approaches the lower limit (red bounding box, Figure 3a) of
the calculated absorbance for the twisted conformation (587
nm). In contrast to the anti-folded isomers, the E and Z forms
of the twisted conformation are theoretically differentiated, with
the Z form predicted to be red-shifted by 0.1 eV (30 nm).
Taken together, UV−vis spectroscopy and excited state
calculations support a preference for the anti-folded con-
formation in solution. Excitation and emission spectra are
shown in Figure 3b, and the quantum yield of E-1/Z-1 in
2). With a decoupling pulse (3610 Hz) at room temperature,
the two doublets at δ 8.26 became two singlets spaced 31.7 Hz
apart. Repeating the heating regimen while decoupling allowed
for clearer observation of coalescence at 45 °C (k
= 70.4
exchange
−1
s ) and fast exchange at 70 °C (see Supporting Information).
From the temperature dependent NMR data, the activation
energy for the E-1/Z-1 isomerization was determined to be
⧧
ΔG = 15.9 kcal/mol.
Having established the solid-state structure as the achiral
anti-folded conformation and the feasibility of a dynamic
isomerization process even below room temperature (≤10 °C),
we focused our attention on the conformational preference in
solution. At this point, all of the data suggested an
interconverting mixture of E/Z isomers in solution. The issue
of twisted versus anti-folded in solution remained, and we
addressed it using a combination of UV−vis spectroscopy and
2
7−31
excited state computational methods.
determined absorbance spectrum for E-1/Z-1 shows a
featureless band in CHCl with a single maximum at λ
The experimentally
CHCl was found to be Φ = 0.088 ± 0.002.
3
15
Xylopyridine A was reported to bind CT-DNA, by
intercalation. We found that the structure reported as
xylopyridine A (E-1/Z-1) did not bind CT-DNA, although
solubility was a major impediment in a variety of neutral-to-
acidic buffers. Treatment of E-1/Z-1 with dimethyl sulfate in
toluene led to water-soluble N-methylpyridinium analogues
E‑6/Z‑6 (Figure 4a), facilitating nucleic acid binding studies
using the equilibrium dialysis method pioneered by Chaires et
=
max
3
−1
−1
3
75 nm (ε = 16 659 ± 86 M cm ). This is in stark contrast
to the reported UV−vis of xylopyridine A, which shows a band
with vibronic structure and maxima at λ = 376, 398 (λ ),
abs
max
1
5
and 425 nm. The possibility of a twisted conformation in
solution was evaluated using excited state calculations to
32
simulate the UV−vis spectrum.
39−41
Using various levels of theory we were able to bound our
experimental result and make a reasonable prediction regarding
the presence of the twisted conformation (Figure 3a,
Supporting Information Table S2). Levels of theory that span
al.
The methylated analogues (E-6/Z-6) did not bind CT-
DNA; however, binding was observed for the human telomeric
sequence (Figure 4b), demonstrating that the anti-folded
diazaxanthylidene architecture can selectively bind quadruplex
forming nucleic acid sequences over B-form DNA. This level of
selectivity is dramatic compared to the commonly used
commercially available quadruplex binder TMPyP4 (cationic
3
3,34
a range of accuracy from overestimating (i.e., CIS)
to
3
3,34
underestimating (i.e., LSDA),
more accurate hybrid methods such as B3LYP,
in addition to putatively
35,36
37
M06, and
C
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Figure 4. (a) Chemical structure of the N-methylpyridinium derivative
of E-1/Z-1. (b) Results of equilibrium dialysis experiments of E-6/Z-6
with B-form DNA and telomeric DNA (5′-AGGG(TTAGGG) -3′).
7
C
b
is the bound ligand concentration, and C is the free ligand
f
concentration.
porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin),
which only shows a 2.5-fold selectivity for binding telomeric
DNA over B-form DNA. Despite considerable debate over the
existence and relevance of quadruplexes in vivo, mounting
evidence is pointing toward quadruplex nucleic acids as an
42,43
emerging oncology target.
Recently, using structure-specific
antibodies as probes, Balsubramanian et al. reported results
consistent with the observation of quadruplexes in human
4
4
cells.
The lack of affinity for B-form DNA can be attributed to the
lack of planarity caused by the overcrowded anti-folded
molecular architecture of E-6/Z-6. Intercalators are generally
4
5
planar polyaromatic molecules as exemplified by cryptolepine,
Figure 5. (a) Crystal structure of E-1, showing a folding angle of 144°
between the phenyl and pyridyl planes of each tricyclic system. (b)
Crystal structure of the intercalator cryptolepine, showing a planar
structure with a height of h = 3.4 Å.
which can effectively stack against base-pairs via π−π stacking
interactions. The local unwinding of the nucleic acid helix at an
intercalation site results in a translation of the base-pairs along
the helix axis a distance equal to the height of an aromatic ring
(
h ≈ 3.4 Å, Figure 5b). The anti-folded alignment of the
nonplanar three-ring systems in E-6/Z-6 results in a molecular
geometry with a height of 5.8 Å, compared to a height of 3.4 Å
for a standard intercalator. Within each three-ring system of the
anti-folded conformer there is an angle of 144° between the
corresponding phenyl and pyridyl planes (Figure 5a). This
conformation lacks a continuous planar π surface that extends
beyond a single six-membered ring, limiting its ability for
effective π−π interactions. The specific stabilizing effects that
account for the binding of E-6/Z-6 with telomeric DNA remain
to be determined. However, such specificity compared to B-
form DNA binding suggests that this scaffold could serve as a
starting point for the design of quadruplex-specific ligands with
a novel architecture that locks out nonspecific intercalative
binding modes. Future studies focused on targeting unique
quadruplex topologies with this new scaffold will be of
considerable interest, given its novel architecture.
conformation in solution and the solid state was determined to
be anti-folded. While the identity of xylopyridine A remains
unresolved, the synthesis of E-1/Z-1 has provided a new
molecular scaffold for investigating structure-specific nucleic
acid binders. The ability to lock out nonspecific B-form DNA
intercalation while maintaining affinity for guanine tetrads will
be key to the field of quadruplex structure-specific nucleic acid
targeting. In depth studies of this new class of conformationally
dynamic structure-specific nucleic acid binding scaffold are
underway and more details will be reported in due course.
EXPERIMENTAL SECTION
■
General Information. n-Butyllithium in hexanes was purchased
from Acros and titrated using diphenylacetic acid recrystallized from
toluene. All salts used for equilibrium dialysis were purchased from
Fisher Bioreagents. The telomeric DNA (5′-AGG GTT AGG GTT
AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG G-3′)
was purchased from Integrated DNA Technologies. All other reagents
were purchased from Sigma Aldrich and used without further
purification. Reactions requiring anhydrous conditions were run
under argon, using Fisher solvents dried via alumina column. Silica
chromatography was done using Silicycle silica gel (55−65 Å pore
diameter). Thin-layer chromatography was performed using Sorbent
Technologies silica plates (250 μm thickness).
CONCLUSIONS
■
In summary, the reported structure of xylopyridine A (E-1) has
been synthesized and found to have a low isomerization barrier,
allowing for equilibration with its Z isomer in solution on an
experimentally relevant time scale (kexchange = 70.4 s at 45 °C).
Using a combination of experiment and theory, the preferred
−1
D
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1H and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, on a Bruker UNI 500 H NMR. High-resolution mass
mL), and extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over Na SO , concentrated in vacuo,
1
2
4
spectra were obtained by Dr. Rakesh Kohli at the University of
Pennsylvania Mass Spectrometry Service Center on a Micromass
AutoSpec electrospray/chemical ionization spectrometer. X-ray
diffraction structure determination was performed by Dr. Patrick
Carroll at the University of Pennsylvania. Infrared spectra were
obtained on a Perkin-Elmer BX FT-IR spectrometer. Ultraviolet
absorption spectrophotometry was performed on a JASCO V-650
spectrophotometer with a PAC-743R multichannel Peltier using
quartz cells with 1 cm cell path lengths. DSC was performed on a TA
Instruments Q 2000 differential scanning calorimeter. Reversed-phase
column chromatography was performed using a Teledyne Isco
CombiFlash Rf system on RediSep Rf Gold C18 columns. High-
performance liquid chromatography analysis was carried out using a
Jasco HPLC instrument equipped with a Phenomenex column (Luna
and run through a short silica plug using 1:1 EtOAc:hexanes to
provide crude 3-chloro-4-cyanopyridine (1.196 g). Forty-two percent
of this crude product was carried on to the next step.
46
Phenol (0.692 g, 7.4 mmol), NaH 60% dispersion in mineral oil
(0.296 g, 7.4 mmol), Cs CO (1.242 g, 3.8 mmol), and DMF (8 mL)
2
3
were stirred under argon at room temperature. When the bubbling
stopped, a solution of the above crude product (0.507 g) in 1 mL of
DMF was added, and then rinsed with 1 mL of fresh DMF. After being
stirred at 50 °C for 2.5 days, the reaction mixture was allowed to cool
to room temperature, quenched with saturated NH Cl, and extracted
4
with DCM. The extract was washed with brine, dried with Na SO ,
2
4
and then concentrated in vacuo to give a brown oil (1.137 g). This oil
was transferred to a beaker using a small amount of DCM. After the
DCM was removed by stirring at 40 °C, 45 g of polyphosphoric acid
was added, and the mixture was stirred at 210 °C overnight. Next, the
solution was cooled to room temperature, poured over ice water, and
neutralized slowly with concentrated NaOH. The resulting mixture
was filtered through Celite and washed with water followed by DCM.
The aqueous layer was extracted with DCM. The combined organic
layers were washed with brine, dried over Na SO , and evaporated
5
u C18(2) 100A; 250 × 4.60 mm, 5 μm). Equilibrium dialysis
experiments were performed in a Thermo Scientific Pierce RED plate
with 8K molecular-weight cutoffs.
All calculations were performed using Gaussian 09. All local minima
were optimized in the gas phase using DFT-B3LYP and M06 methods
with the 6-311+G(2d,p) basis set; they were then confirmed to be
stable and to have no imaginary frequencies by stability and frequency
calculations, respectively, using the same basis set. All UV−vis
calculations were performed using CIS, TD-ωB97X-D, TD-B3LYP,
TD-PBEPBE, TD-M06, and TD-LSDA methods with the same basis
set in chloroform.
2
4
4
8
under reduced pressure to afford 3-azaxanthone 4 as a white solid
(0.463 g, 2.3 mmol, 56% over three steps).
3-Aza-9H-xanthene-9-thione (5). A mixture of 3-azaxanthone 4
(1.004 g, 5.1 mmol) and Lawesson’s reagent (1.257 g, 3.1 mmol) in a
250 mL round-bottom flask was dried at 40 °C under high vacuum for
1 h, and then the flask was back-filled with argon. The mixture was
taken up in 160 mL of alumina-dried toluene, heated to 80 °C, and
stirred for 18 h. The reaction was concentrated in vacuo, and the crude
product was purified by silica gel chromatography (15:85 EtOAc:hex-
anes) to provide thione 5 as a green solid (1.052 g, 4.9 mmol, 97%).
4
-Cyano-3-phenoxypyridine (3). In a 50 mL round-bottom
flask, phenol (0.809 g, 8.6 mmol) and cesium carbonate (1.645 g, 5.0
mmol) were taken up in DMF (10 mL) and treated with neat sodium
hydride (0.339 g, 8.5 mmol). After hydrogen evolution ceased, 3-
46
chloro-4-cyanopyridine (0.583 g, 4.2 mmol) was added neat, and the
reaction was stirred at 50 °C for 36 h. Solvent was removed under
vacuum, and the crude product was transferred to a separatory funnel
−1
1
IR (neat, cm ): 1415, 1330. H NMR (500 MHz, CDCl ): δ 9.02
3
(1H, s), 8.62 (1H, d, J = 8 Hz), 8.55 (1H, d, J = 5 Hz), 8.35 (1H, d, J =
5 Hz), 7.8 (1H, t, J = 8 Hz), 7.52 (1H, t, J = 4 Hz), 7.39 (1H, t, J = 8
with 25 mL of water and 25 mL CHCl . The aqueous layer was
3
Hz). 13C NMR (126.9 MHz, CDCl
): δ 176.6, 156.2, 151.2, 144.5,
142.7, 136.2, 127.1, 126.4, 125.0, 122.4, 118.7, 118.5. HRMS (m/z):
extracted with CHCl (three times, 25 mL). The organic layers were
3
3
combined, washed with 40 mL of 2 N NaOH, dried over MgSO4,
+
concentrated in vacuo, and purified by silica gel chromatography (1:4
[M + H] calcd for C12H NOS, 214.0327; found, 214.0327.
7
47
EtOAc:hexanes) to provide 4-cyano-3-phenoxypyridine 3 (0.604 g,
Synthetic Procedure for (E/Z)-3,3′-Diazaxanthylidene (1).
Copper powder (5.133 g, 80.8 mmol) was suspended in toluene (80
mL), 75 mL of which was distilled off to remove water. The distillation
apparatus was replaced with a reflux condenser, and the apparatus was
flushed with argon before a solution of thione 5 (0.516 g, 2.4 mmol) in
20 mL of toluene was cannulated into the copper suspension. The
resulting mixture was heated to 120 °C and stirred for 20 h. The
mixture was then gravity filtered, rinsing the copper with 80 °C
1
3
.1 mmol, 73%). H NMR (500 MHz, CDCl ): δ 8.46 (1H, d, J = 5
3
Hz), 8.33 (1H, s), 7.53 (1H, d, J = 5 Hz), 7.46 (2H, dd, J = 7, 9 Hz),
.28 (1H, t, J = 7 Hz), 7.12 (2H, d, J = 9 Hz).
-Aza-9H-xanthen-9-one (4), Procedure I. Polyphosphoric acid
9.701 g, excess) was added to a beaker containing 4-cyano-3-
7
3
(
47
phenoxypyridine 3 (0.202 g, 1.0 mmol), and the reaction was stirred
at 225 °C for 6 h. The reaction was allowed to cool before being
diluted with 200 mL of water and slowly basified to pH 7 with solid
NaOH. The mixture was transferred to a separatory funnel with 50 mL
of DCM, and the aqueous layer was extracted with DCM (three times,
toluene (100 mL) and boiling CHCl
were combined, concentrated to dryness, dissolved in the minimum
volume of boiling CHCl , cooled to room temperature, treated with
Et O (45 mL), and placed in a 4 °C refrigerator for 16 h. Yellow
crystalline 3,3′-diazaxanthylidene (E-1/Z-1) (0.198 g, 0.6 mmol, 45%)
was recovered via Buchner funnel filtration and rinsing with Et O.
(100 mL). The organic fractions
3
3
5
0 mL). The organic layers were combined, dried over MgSO4,
2
concentrated in vacuo, and purified by silica gel chromatography (3:7
48
EtOAc:hexanes) to provide 3-azaxanthone 4 (0.1679 g, 0.85 mmol,
2
−1
1
8
3%). IR (neat, cm ): 1682, 1417, 761. H NMR (500 MHz,
Purification of the mother liquor by silica gel chromatography (3:2
EtOAc:hexanes) provided additional 3,3′-diazaxanthylidene 1 (0.134 g,
CDCl ): δ 9.01 (1H, s), 8.62 (1H, d, J = 5 Hz), 8.29 (1H, dd, J = 1.5, 8
3
−1
Hz), 8.06 (1H, d, J = 5 Hz), 7.82−7.72 (1H, m), 7.575 (1H, d, J = 8.5
0.4 mmol, 30%) as a yellow powder. IR (neat, cm ): 1410, 1205, 756.
Hz), 7.40 (1H, t, J = 7.5 Hz). 13C NMR (126.9 MHz, CDCl ): δ
1
H NMR (500 MHz, acetone-d ): δ 8.69 and 8.68 (4H, overlapping
3
6
1
1
76.6, 156.2, 151.2, 144.5, 142.7, 136.2, 127.1, 126.4, 125.0, 122.4,
singlets), 8.23 and 8.20 (4H, overlapping doublets, J = 5 and 5 Hz),
+
1
18.8, 118.5. HRMS (m/z): [M + H] calcd for C H NO , 198.0555;
7.46−7.40 (8H, m), 7.26−6.99 (12H, m). H NMR (500 MHz,
12
7
2
found, 198.0559.
CDCl ): δ 8.71 and 8.70 (4H, overlapping singlets), 8.21 and 8.17
3
3
-Aza-9H-xanthen-9-one (4), Procedure II. A round-bottom
(4H, overlapping doublets, J = 5 and 5 Hz), 7.38−7.29 (8H, m), 7.19−
1
flask was charged with 2,2,6,6-tetramethylpiperidine (3.50 mL, 20.7
mmol) and THF (40 mL). The solution was cooled to −30 °C under
argon and then treated with 2.38 M n-butyllithium in hexanes (8.34
mL, 19.8 mmol). The solution was allowed to warm to 0 °C and stir
for 30 min before being cooled back down to −78 °C. A solution of 4-
cyanopyridine 2 (1.030 g, 9.9 mmol) in THF (20 mL) was added over
5 min via syringe pump. After 30 min of stirring at −78 °C, a solution
of hexachloroethane (5.000 g, 21.1 mmol) in THF (10 mL) was added
over 15 min. After 30 min of stirring at −78 °C, the solution was
warmed to room temperature, quenched with saturated NH Cl (40
7.13 (4H, m), 7.07- 7.04 (4 H, m), 7.00−6.92 (4H, m). H NMR (500
MHz, DMF-d ): δ 8.75 (4H, bs), 8.29−8.20 (4H, m), 7.52−7.42 (8H,
7
1
m), 7.28−7.00 (12H, m). H NMR (500 MHz, 3:1 DMF-
d :F CCO D): δ 9.35 (4H, bs), 6.9 (4H, dd, J = 5.5, 11 Hz), 8.13
7
3
2
(2H, d, J = 5.5 Hz), 7.87 (2H, d, J = 6 Hz), 7.59−7.49 (8H, m), 7.45
1
3
(2H, d, J = 8 Hz), 7.29 (2H, d, J = 8 Hz), 7.18−7.12 (4H, m).
C
1
NMR (126.9 MHz, CDCl ): δ 162.2, 162.0, 154.8, 154.6, 138.7, 138.2,
3
138.2, 137.7, 135.2, 134.1, 131.6, 131.3, 128.3, 128.0, 125.1, 124.7+,
124.5, 122.9, 122.9, 121.3, 120.8, 18.0, 117.8. HRMS (m/z): [M + H]
calcd for C H N O , 363.1134; found, 214.0327.
4
24 14
2
2
E
dx.doi.org/10.1021/ja404737q | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
E/Z)-N-Methyl-3,3′-diazaxanthylidene (6). In a two-necked
and Joseph Subotnik for helpful discussions regarding excited
state calculations. This work was supported by funding from
the University of Pennsylvania. We thank the Vietnam
Education Foundation for funding (VEF Fellowship to
M.N.T.) and the NSF for support of the X-ray diffractometer
round-bottom flask, 3,3′-diazaxanthylidene (0.041 g, 0.11 mmol) was
suspended in toluene (20 mL). Next, a solution of dimethyl sulfate 1%
in toluene (0.53 mL, 0.055 mmol) was slowly added. After a 12 h
reflux, the mixture was charged with another batch of dimethyl sulfate
1
% in toluene (0.53 mL, 0.055 mmol) and refluxed for 24 h before
(
Grant CHE-0840438).
cooling to room temperature. The mixture was filtered, and the solid
was washed several times with toluene and then dissolved in water.
The desired product was isolated using reversed-phase column
chromatography with 0.1% TFA/water and acetonitrile as eluents
0.030 g, 0.06 mmol, 55%). IR (neat, cm ): 1693, 1200. H NMR
500 MHz, D O): δ 9.06 and 9.03 (2H, overlapping singlets), 8.91
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ASSOCIATED CONTENT
Supporting Information
■
*
S
Characterization data for all new compounds, X-ray crystallo-
graphic data (CIF), and computational details. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.,
Corresponding Author
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
revision B.01; Gaussian, Inc.: Wallingford, CT, 2010.
dcheno@sas.upenn.edu
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