Ratiometric pH responsive fluorescent probes operative on ESIPT

Article abstract of DOI:10.1016/j.tet.2013.05.023

A series of C-8′-oxime-appended spirobenzopyrans 4a-4d were synthesized and developed as fluorescent pH ratiometric probes operative in HEPES/ACN (8:2, v/v) buffer solutions. Acidochromic conversion of spirobenzopyran to merocyanine open form can facilita

Full text of DOI:10.1016/j.tet.2013.05.023

Tetrahedron 69 (2013) 5874e5879
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Ratiometric pH responsive fluorescent probes operative on ESIPT
,
,
a
Yim-Pan Chan a, Li Fan a b, Qihua You a, Wing-Hong Chan a , Albert W.M. Lee ,
*
Shaomin Shuang b
a Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region
b Center of Environmental Science and Engineering Research, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
PR China
a r t i c l e i n f o
a b s t r a c t
A series of C-80-oxime-appended spirobenzopyrans 4ae4d were synthesized and developed as fluo-
rescent pH ratiometric probes operative in HEPES/ACN (8:2, v/v) buffer solutions. Acidochromic con-
version of spirobenzopyran to merocyanine open form can facilitate the subsequent enoleketo
tautomerization giving dual emissive peaks via the excited state intramolecular proton transfer (ESIPT)
Article history:
Received 22 January 2013
Received in revised form 22 April 2013
Accepted 10 May 2013
Available online 16 May 2013
mechanism. The pH titrations of 4a show a 68-fold increase in ratiometric intensities ratio (I645
I522 nm) when the pH was switched from 8.0 to 4.0 with a pKa value of 5.90.
/
nm
Keywords:
Spirobenzopyran
pH sensing
Ó 2013 Elsevier Ltd. All rights reserved.
Ratiometric fluorescent probe
ESIPT
1. Introduction
non-fluoresced spirobenzopyran close form to fluoresced mer-
ocyanine open form contributed to the basis for the turn-on sensor
development. It is alsowell known that such a transformation can be
achieved by acidification of spirobenzopyran derivatives.7
The present report is directed toward the synthesis and fluori-
metric characterization of spirobenzopyran derivatives 4ae4d
possessing tunable pKa values close to the physiological pH range
that can detect the pH changes of aqueous solutions. Operating on
the ESIPT mechanism, the probes in response to pH variations
afforded ratiometric fluorescent signals originated from the enol-
and keto-tautomer, respectively.
Owing to their intrinsic simplicity and selectivity, fluorescent
probes are versatile tools for visualizing and detecting metal cations,
anions, and biologically relevant small organic molecules.1e3 Mea-
surement of pH by fluorescence-based techniques has important
implications in analytical and biological chemistry. Development of
fluorescent pH sensing probes for both imaging and sensing appli-
cations has received much attention in the current literature.4
Probes characterized by their ratiometric and ‘offeon’ signal out-
puts in response to physiological pH variations are widely sought
after sensing devices. A handful of detection schemes exploiting
proton binding induced perturbations of de-excitation pathways
such as photo-induced electron transfer (PET),4a,b intramolecular
charge transfer (ICT),4e Forster resonance energy transfer (FRET)4c,d
have been used for the development of pH sensing probes.
Recently, excited state intramolecular proton transfer (ESIPT) has
emerged as an effective fluorescent signal transduction mechanism
for designing novel ratiometric fluorescent probes. For instance,
operating on ESIPT, fluorescent probes for Hg(II), pyrophosphate,
and cysteine have been established in the literature.5 In recent years,
spirobenzopyrans were chosen by us as the molecular scaffold for
developing novel fluorescent chemosensors for Cu2þ, Zn2þ, pyro-
phosphate, and glutathione.6 Analyte induced conversion of the
2. Results and discussion
Acidochromic ring opening of spirobenzopyran affords the cor-
responding merocyanine bearing
a phenolic moiety. By in-
corporating an oxime moiety onto the C-80 of the spirobenzopyran
skeleton, the merocyanine derivatives formed under acidic condi-
tions will possess a molecular platform, which can undergo intra-
molecular proton transfer from the phenolic oxygen to the oxime
nitrogen in the photo-excited state as shown in Scheme 1.
Our rational design of spirobenzopyran-based fluorescent pH
probes possesses two attractive features. The pH responsive char-
acteristics of the probes can be tuned by incorporating of different
substituents onto the benzene ring. Upon acidification and excita-
tion, the enol produced at its excited state could undergo tauto-
merization gives rise to an emission with large Stokes’ shift. In
* Corresponding author. Tel.: þ852 34117076; fax: þ852 34117348; e-mail ad-
dress: whchan@hkbu.edu.hk (W.-H. Chan).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.023

Y.-P. Chan et al. / Tetrahedron 69 (2013) 5874e5879
5875
R
R
H3O+
ESIPT
N
O
N
O
R
N
O
H
HN
8'
N
OH
OH
OH-
keto phototautomer
N
HO
OH-
R
N
O
H
N
O
Scheme 1. Acid induced conversion of spirobenzopyran to merocyanine undergoing ESIPT.
essence, the dual normal and ESIPT de-excitation pathway would
give two emissive peaks as ratiometric output signals.
centered at 385 and 425 nm as shown in Fig.1A. In alkali conditions,
a new absorption peak at 550 nm with low intensity emerges with
a clear isosbestic point at 500 nm. The observed red-shifted ab-
sorption peak could be due to the intramolecular charge transfer of
the ring open merocyanine form taking place in the ground state. It
is noteworthy that, in contrast to other substituted spirobenzopyr-
ans, the presence of the oxime hydroxyl moiety may stabilize the
phenoxyl group preventing its cyclization back to the close form. To
substantiate this presumption, a control compound 5 was synthe-
sized by alkylating the oxime hydroxyl group with benzyl chloride
(Fig. S1). The UVevis spectra of 5 at different pH revealed that the
merocyanine open form of 5 underwent ring closure to the spi-
robenzopyran close form at alkali solutions (Fig. S2).
The syntheses of 60-subsituted 80-oxime-appended spi-
robenzopyrans 4ae4d were carried out efficiently in a three-step
reaction sequence starting from the respective 4-substitued phenol
shown in Scheme 2. Using the literature protocol, clean diformyla-
tion of p-substituted phenols 1ae1d was accomplished by reaction
with 2 equiv of hexamethylenetetramine in refluxing trifluoroacetic
acid, affording the corresponding dialdehydes 2ae2d, respectively.8
Subsequent treatment of the dialdehydes with 1 equiv of hydrox-
ylamine hydrochloride salt in the presence of 1 equiv of NaOH gave
the corresponding mono-oximes 3ae3d, respectively, in 31e51%
yield. Good yield of 4ae4d was obtained by heating the mono-
oximes separately with 1.2 equiv of N-methyl-2,3,3-
trimethylindolenine iodide and morpholine in absolute ethanol.9
The fluorescence spectra of 4a excited at 425 nm in HEPES/ACN
buffer solutions of pH 3.0e9.0 were recorded. In pH¼9.0, 4a ex-
N
R
R
R
2
N
NH2OH.HCl
N
N
OHC
OHC
CHO
OH
2 a- d
1 equiv NaOH
31 - 51%
TFA
OH
N
OH
OH
1a - d
3 a - d
a
t-butyl
OCH3
Br
R =
N
I
b
c
d
N
O
R
N
O
O
5
O
4 a - d
50 - 82%
N
N
phenyl
HO
N
H
Scheme 2. Synthetic route to the fluorescent pH sensing probes and the structure of control compound 5.
With the fluorescent sensory materials in hand, we explored
hibits only one emissive peak at 522 nm. With an increase in pH, its
fluorescence intensity at 522 nm was reduced and a new emission
band at 645 nm emerged concomitantly. The band at higher energy
could be attributed to the emission from hydrogen bonded enol
tautomer. The large Stokes’ shifted emission band at lem¼645 nm
can conceivably be assigned to the keto-tautomer form in excited
state from the enol-tautomer (Scheme 1). The phototautomer
possessing short life time (i.e., in picosecond) emits long wave light
and back to the ground state.10 Apparently, the pH variation triggers
the display of ratiometric fluorescence signal changes from 4a. A
clear isoemission point is apparent at 590 nm, which indicated the
their use as fluorescent pH sensing probes. On the outset of the in-
vestigation, for practical reason, we chose 8:2 aqueous buffered
acetonitrile solution as the solvent for studying the pH responsive
properties of the probes. The spectroscopic properties of 4a were
first selected to be examined under different pH conditions. The
acidochromic interconversion of the spirobenzopyran close form of
4a to the extended conjugated merocyanine ring open form was
evidenced from its UVevis absorption spectra obtained from dif-
ferent HEPES buffer solutions. Under strong acidic conditions, 4a
exhibits two typical
pep partially overlapped absorption bands
*

5876
Y.-P. Chan et al. / Tetrahedron 69 (2013) 5874e5879
1.2
1.0
0.8
0.6
0.4
0.2
0.0
250
A
B
pH 3.0
pH 4.0
425 nm
pH 3.0
pH 4.0
pH 4.3
pH 4.7
pH 5.0
pH 5.3
pH 5.5
pH 5.9
pH 6.3
pH 6.7
pH 7.1
pH 7.5
pH 8.0
pH 9.0
645 nm
pH 4.3
200
pH 4.7
pH 5.0
pH 5.3
150
pH 5.5
522 nm
pH 5.9
pH 6.3
pH 6.7
100
pH 7.1
pH 7.5
pH 8.0
pH 9.0
50
550 nm
0
300
400
500
600
700
450 500 550 600 650 700 750 800 850
Wavelength (nm)
Wavelength (nm)
Fig. 1. UVevis absorption (A) and fluorescence emission spectra (B) of 4a (50
m
M) at different pH values in HEPES buffer/ACN (8:2, v/v) with lex¼425 nm.
equilibrium between the keto and the enol form. A plot of the ratios
of the emission intensities of 4a at 645 and 522 nm against the pH
of the solutions is shown in Fig. 2, displaying the typical sigmoidal
relationship between the two parameters (R2¼0.995). This
intensities ratio decreased from 13.48 to 0.20 associated with pH
change from 3.0 to 9.0, giving a huge value change of 68 between
two plateaus of the pH titration curve. Non-linear curve fitting
using Boltzmann equation, on the basis of the plot of fluorescence
intensities ratios against pH, the pKa of the probe was calculated to
be 5.90.4d The typical application range of 4a can be estimated to be
4.0e8.0. Additionally, the probe displayed a fast response time
toward the pH change of the solutions. As shown in Fig. S3, stable
and steady fluorescent readings can be obtained within 6 min.
To define the application scope of this pH probe, its response to
other common metal ions should be examined. To investigate this
phenomenon, metal ion selectivity assays were conducted at both
pH 4.0 and 7.0. Upon addition of 2400 equiv (120 mM) of Naþ, Kþ to
4a solution, no change in its fluorescence was observed at both pH
values. Furthermore, adding 6 equiv of other 12 common metal ions
14
12
10
8
6
(0.3 mM), i.e., Liþ, Ca2þ, Mg2þ, Fe2þ, Fe3þ, Zn2þ, Co2þ, Ni2þ, Cd2þ
,
4
Hg2þ, Pb2þ, Agþ separately into 4a solution did not cause any ad-
verse effect on the ratios of emission intensities of the probe, and
the ratios remained almost constant at pH 4.0 and 7.0, respectively
(Fig. 3). The interference studies further revealed that only Cu2þ
exhibited substantial quenching effect on the fluorescence output
signals of 4a, irrespective of the pH of the solution. The interference
results imply that 4a can selectively measure pH in the presence of
various metal ions usually present in biological systems. Further-
more, the significant interference effect caused by Cu2þcan be
2
0
3
4
5
6
7
8
9
pH
Fig. 2. Plot of the emission intensities ratios of R at I645 nm/I522
HEPES buffer/ACN (8:2, v/v) with lex¼425 nm.
to pH variation in
nm
B
A
pH 4.00
pH 7.00
14
1.2
1.0
0.8
0.6
0.4
0.2
0.0
12
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Fig. 3. Metal ion selectivity profiles of 4ademission ratios (I645 nm/I522 nm) of 4a (50
m
M) in 80% HEPES/ACN [50 mM, pH¼4.00 (A) and pH¼7.00 (B)]. lex¼425 nm, slit¼10/10 nm. 1,
free; 2, Liþ (0.3 mM); 3, Naþ (120 mM); 4, Kþ (120 mM); 5, Ca2þ (0.3 mM); 6, Mg2þ (0.3 mM); 7, Cu2þ (0.3 mM); 8, Fe3þ (0.3 mM); 9, Fe2þ (0.3 mM); 10, Zn2þ (0.3 mM); 11, Co2þ
(0.3 mM); 12, Ni2þ (0.3 mM); 13, Cd2þ (0.3 mM); 14, Agþ (0.3 mM); 15, Hg2þ (0.3 mM); and 16, Pb2þ (0.3 mM).

Y.-P. Chan et al. / Tetrahedron 69 (2013) 5874e5879
5877
conveniently removed by using o-ethylxanthic acid as the screen-
A 100
90
pH 3.0
ing agent. As shown in Fig. S4, the quenching effect on the probe
caused by Cu2þ (0.3 mM) in both pH 4.00 and 7.00 buffered solu-
tions can be eliminated by the addition of a same amount of o-
ethylxanthic acid.
pH 4.0
pH 4.3
pH 4.7
pH 5.0
pH 5.3
pH 5.5
pH 5.9
pH 6.3
pH 6.7
pH 7.1
pH 7.5
pH 8.0
pH 9.0
686 nm
80
70
It is our intention to design and synthesize a series of potential
pH fluorescent probes possessing tunable pKa values. The nature
of substituents appended at the C-60 position of spirobenzopyrans
could affect the photophysical properties of the materials.
Therefore, using the same synthetic protocol outlined in Scheme
1, spirobenzopyrans 4b, 4c, and 4d were synthesized for com-
parative investigation. Starting from the corresponding 4-
substituted phenol, all these three compounds can be obtained
in moderate overall yield. Their structures were fully character-
ized by NMR and HRMS spectroscopy. Their optical characteristics
in response to pH change were established by UVevis and fluo-
rescence spectroscopy. UVevis absorption spectral changes of 4b
and 4c in aqueous ACN solution at different pH values are shown
in Fig. S5. To assess their applicability as pH fluorescent sensing
probes, their fluorescent spectra in 8:2 HEPES buffer and ACN (v/
v) at different pH were measured and are shown in Fig. 4. To
certain extent, the photophysical properties of these three ana-
logs of 4a are very similar. Dual fluorescence outputs were ob-
served at different pH solutions. Apparently, the rapid process of
enoleketo tautomerization as a result of ESIPT took place for all
these materials under acidic conditions. Evidently, compounds
4ae4d are suitable materials for the development of ratiometric
pH fluorescent probes.
Careful examination of the fluorescence data of these materials
revealed that different substituents appended at the C-60 of spi-
robenzopyran scaffold would cause some subtle changes of pho-
tophysical properties of the resultant compounds. However, the
ratio between the dual fluorescence intensities of the four spi-
robenzopyrans correlated nicely with the pH values of the solutions
(Fig. 5). Acting as ratiometric pH fluorescent probes, the pH dy-
namic working range and the response sensitivity of the probes are
structurally dependent. Compound 4d with a C-60 substituted
phenyl group exhibited the highest sensitivity to the pH variation of
the solution. Under pH 4.0 and 9.0, a large difference of 175-fold in
intensities ratio was recorded for 4d. On the other hand, 4b with
a C-60 substituted electron donating methoxy group displays the
smallest separation in intensities ratio under the acidic and basic
conditions.
60
50
557 nm
40
30
20
10
0
500
550
600
650
700
750
800
850
Wavelength (nm)
B
pH 3.0
pH 4.0
pH 4.3
pH 4.7
pH 5.0
pH 5.3
pH 5.6
pH 5.9
pH 6.3
pH 6.7
pH 7.1
pH 7.5
pH 8.0
pH 9.0
350
650 nm
504 nm
300
250
200
150
100
50
0
450
500
550
600
650
700
750
800
850
Wavelength (nm)
300
250
200
150
100
50
C
pH 3.0
pH 4.0
pH 4.3
pH 4.7
pH 5.0
pH 5.3
pH 5.6
pH 5.9
pH 6.3
pH 6.7
pH 7.1
pH 7.5
pH 8.0
pH 9.0
673 nm
For clear comparison of the photophysical properties of the
materials, relevant properties of this series of compounds are
compiled in Table 1. The exact position of the emission bands of the
probes is strongly substituent-dependent. The presence of C-60-
methoxy group in the spirobenzopyran induced the greatest red-
shift for both emissive peaks. On the basis of curve fitting, the
pKa found for these four probes is distinctive different ranging from
5.26 to 6.32. The phenyl group is most effective to enhance the
acidity of the probe while strong electronic donating methoxy
group reduced considerably the acidity of the probe. According to
the quantum yield measurement, all synthesized materials are only
modestly fluoresced under both acidic and basic aqueous
conditions.
524 nm
0
450
500
550
600
650
700
750
800
850
Wavelength (nm)
Fig. 4. The fluorescence emission spectra of (A) 4b, (B) 4c and (C) 4d; (all in 50
different pH values in HEPES buffer/ACN (8:2, v/v) with lex¼425 nm.
mM) at
3. Conclusion
In summary, we have successfully designed and synthesized
a series of C-60 substituted C-80 oxime-appended spirobenzopyrans.
The dual fluorescence outputs of all these materials were demon-
strated to be pH dependent in aqueous solutions. The pKa of the
probes could be tuned by changing the substituent pattern at C-60
of the molecules. The cross interference caused by common metal
ions except Cu2þ to the pH probes is negligible.
4. Experimental section
4.1. General
The melting point was determined with a MEL-TEMPII melting
point apparatus (uncorrected). 1H NMR and 13C NMR spectra
were recorded on Bruker Advance-III 400 spectrometer (at 400 and

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Y.-P. Chan et al. / Tetrahedron 69 (2013) 5874e5879
4.3. 5-(tert-Butyl)-2-hydroxy-3-((hydroxyimino)methyl)benz-
aldehyde (3a)
4a pKa = 5.90
4b pKa = 6.32
4c pKa = 5.50
4d pKa = 5.26
25
20
15
10
5
A
solution of 5-(tert-butyl)-2-hydroxyisophthalaldehyde
(1.085 g, 5.36 mmol) in ethanol (50 mL) was mixed with a solu-
tion of hydroxylamine hydrochloride (366 mg, 5.36 mmol) and
sodium hydroxide (25 mg). The reaction mixture was heated to
60 ꢀC for overnight. After being cooled to room temperature, part of
solvent was removed under reduced pressure. Then the reaction
mixture was diluted with water and extracted with ethyl acetate
(3ꢁ50 mL). The combined organic layers were dried by anhydrous
sodium sulfate and filtered. The filtrate was evaporated under
vacuum to afford a crude residue that was purified by column
chromatography (n-hexane/ethyl acetate 50:1) to yield product 3a
0
(441 mg, 38% yield) as pale yellow oil. H NMR (CDCl3-d1)
1.33 (s, 9H), 7.68 (d,1H, J¼2.4 Hz), 7.81 (d, 1H, J¼2.4 Hz), 8.44 (s, 1H),
9.19 (br s, 1H), 10.10 (s, 1H), 11.09 (s, 1H). C NMR (CDCl3-d1) (ppm):
d (ppm):
3
4
5
6
7
8
9
pH
d
31.2, 34.2, 119.3, 121.5, 130.2, 132.5, 142.9, 147.8, 157.8, 194.6.
Compounds 3b, 3c, and 3d were prepared using the same
procedure.
Fig. 5. Plots of the emission intensities ratios (i.e., Iketo/Ienol) of different probes to pH
variation in HEPES buffer/ACN (8:2, v/v) with lex¼425 nm.
Table 1
Photophysical properties of the spirobenzopyran derivatives in HEPES buffer/ACN (8:2, v/v) solutions
(104 Mꢂ1 cmꢂ1
)
lem (nm)
pKa
Intensities ratioa
Rmax/Rmin
w68
Q.Y.c
F
b
Probe
labs (nm)
3
4a
384,
1.681
1.884
0.135
2.190
2.045
0.504
1.849
0.504
0.938
0.841
0.345
522 (pH 9.0)
645 (pH 3.0)
5.90
0.20 (pH 9.0)
13.48 (pH 3.0)
0.04 (pH 3.0)
0.02 (pH 9.0)
425 (pH 3.0)
550 (pH 9.0)
375,
452 (pH 3.0)
588 (pH 9.0)
420 (br) (pH 3.0)
550 (pH 9.0)
375,
4b
557 (pH 9.0)
686 (pH 3.0)
6.32
0.10 (pH 9.0)
6.11 (pH 3.0)
w61
0.007 (pH 3.0)
0.002 (pH 9.0)
4c
504 (pH 9.0)
650 (pH 3.0)
524 (pH 9.0)
673 (pH 3.0)
5.50
5.26
0.11 (pH 9.0)
15.76 (pH 3.0)
0.13 (pH 9.0)
22.75 (pH 3.0)
w143
w175
0.07 (pH 3.0)
0.03 (pH 9.0)
0.05 (pH 3.0)
0.02 (pH 9.0)
4d
430 (pH 3.0)
555 (pH 9.0)
a
Intensities ratio of the emission peak with longer wavelength versus the peak with shorter wavelength.
b
c
Rmax¼intensities ratio at pH 3.0; Rmin¼intensities ratio at pH 9.0.
Fluorescence quantum yield (Q.Y.) was calculated relative to fluorescein (
F
¼0.91 in 0.1 M NaOH).
100 MHz, respectively) in CDCl3. High-resolution mass spectra were
obtained on a Bruker Autoflex mass spectrometer (MALDI-TOF).
Fluorescent emission spectra and UVevis spectra were collected on
a PTI luminescence lifetime spectrometer and a Cary UV-100
spectrometer, respectively. Unless specified, all fine chemicals
were used as received.
Compound 3b (obtained as pale yellow oil in 43% yield): H NMR
(CDCl3-d1)
(ppm): 3.83 (s, 3H), 7.21 (d, 1H, J¼3.2 Hz), 7.37 (d, 1H,
d
J¼3.2 Hz) 8.40 (s, 1H), 10.13 (s, 1H), 10.61 (s, 1H). C NMR (CDCl3-d1)
d
(ppm): 56.4, 116.4, 120.5, 121.4, 122.2, 125.1, 152.5, 154.4, 193.3.
Compound 3c (obtained as pale yellow oil in 51% yield): H NMR
(CDCl3-d1)
d
(ppm): 7.80 (d, J¼2.5 Hz), 7.90 (d, J¼2.5 Hz), 8.27 (s,
1H), 10.10 (s, 1H), 11.02 (br s, 1H). C NMR (CDCl3-d1)
d (ppm): 117.1,
122.0, 132.3, 137.8, 138.6, 161.6, 148.8, 191.8.
Compound 3d (obtained as pale yellow oil in 50% yield): H NMR
(CDCl3-d1)
(ppm): 7.37 (t, 1H, J¼7.3 Hz), 7.46(t, 2H, J¼7.8 Hz), 7.57
(d, 2H, J¼7.1 Hz), 7.92 (d, 1H, J¼2.3 Hz), 8.03 (d, 2H, J¼1.8 Hz), 8.50
(s, 1H), 10.22 (s, 1H), 11.13 (br s, 1H). C NMR (MeOD-d4) (ppm):
121.7,124.3,127.6,128.6,129.9,130.1,134.3,134.5,140.4,149.1,160.4,
193.6.
4.2. 2,6-Diformyl-4-Phenylphenol (2d)
d
4-Phenylphenol (528 mg, 3.10 mmol) was reacted with
2 equiv hexamethylenetetramine (958 mg, 6.83 mmol) in tri-
fluoroactetic acid (10 mL). The reaction mixture was refluxed at
150ꢀ C for 24 h under nitrogen atmosphere. Then the mixture was
cooled to room temperature and stirred with 4 M HCl (10 mL) for
30 min. The mixture was extracted by dichloromethane (3ꢁ15 mL).
The combined organic layers were washed successively by dilute
hydrochloric acid, water, and saturated brine solution, followed by
drying with anhydrous sodium sulfate. The organic solution was
filtered and evaporated under vacuum to afford a crude residue that
was purified by column chromatography (n-hexane/ethyl acetate
20:1) to yield pure 2d (219 mg, 26.5% yield). H NMR (400 MHz,
CDCl3-d1) 7.40, (tt, 1H, J¼7.4, 1.3 Hz), 7.47 (t, 2H, J¼7.2 Hz), 7.58 (dd,
2H, J¼7.5, 0.6 Hz), 8.18 (s, 2H), 10.31 (s, 2H), 11.63 (s, 1H). Dia-
ldehydes 2ae2c were synthesized according to the literature
procedure.8
d
4.4. 6-(tert-Butyl)-10,30,30-trimethylspiro[chromene-2,20-indo-
line]-8-carbaldehyde oxime (4a)
To a solution of 5-(tert-butyl)-2-hydroxy-3-((hydroxyimino)
methyl)benzaldehyde (20 mg, 0.09 mmol) and N-methyl-2,3,3-
trimethylindolenine iodide (35 mg, 0.10 mmol) in anhydrous eth-
anol (10 mL), morpholine (2 mL) was added dropwise. The reaction
mixture was refluxed at 60 ꢀC for overnight. After being cooled to
room temperature, part of solvent was removed under reduced
pressure. Then the reaction mixture was diluted with water and
extracted with ethyl acetate (3ꢁ10 mL). The combined organic

Y.-P. Chan et al. / Tetrahedron 69 (2013) 5874e5879
5879
layers were dried by anhydrous sodium sulfate and filtered. The
filtrate was evaporated under vacuum to afford a crude residue,
which was purified by column chromatography (n-hexane/ethyl
acetate 100:1) to yield product 4a as purple oil (28 mg, 82% yield). H
397.1910; found: 397.1929. HRMS (MALDI-TOF) for C26H25N2O2
calcd 397.1910 (MþHþ), found, 397.1929.
Acknowledgements
NMR (CDCl3-d1) d (ppm): 1.16 (s, 3H), 1.28 (s, 12H), 2.71 (s, 3H), 5.70
(d, 1H, J¼10.2 Hz), 6.50 (d, 1H, J¼7.7 Hz), 6.81 (dd, 1H, J¼7.2 Hz),
6.83 (d, 1H, J¼10.2 Hz), 7.04 (d, 1H, J¼7.1 Hz), 7.09 (d, 1H, J¼1.9 Hz),
7.15 (dd, 1H, J¼7.5, 7.7 Hz), 7.59 (d, 1H, J¼1.9 Hz), 7.97 (br s, 1H), 8.18
The work was supported by a grant from the Research Com-
mittee of Hong Kong Baptist University (FRG2/11-12/121).
(s, 1H). C NMR (CDCl3-d1)
d (ppm): 20.5, 25.9, 28.9, 31.4, 34.1, 51.8,
Supplementary data
104.9,106.8,117.2,118.8,119.3,120.0,121.5,122.5,125.8,127.6,129.4,
136.4, 142.7, 145.6, 147.9, 150.4. HRMS (MALDI-TOF) for C24H28N2O2
calcd 377.2223 (MþHþ), found, 377.2230.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.05.023.
Using the same procedure, 4b, 4c, and 4d were prepared.
Compound 4b (as purple oil in 43% yield): H NMR (CDCl3-d1)
References and notes
d
(ppm): 1.16 (s, 3H), 1.28 (s, 3H), 2.71 (s, 3H), 3.76 (s, 3H), 5.76 (d,
1H, J¼10.2 Hz), 6.50 (d, 1H, J¼7.7 Hz), 6.69 (d, 1H, J¼3.0 Hz), 6.81 (d,
1. (a) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Chem. Rev. 2012, 112,
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1H, J¼10.2 Hz), 6.84 (d, 1H, J¼3.0 Hz), 7.04 (d, 1H, J¼6.8 Hz), 7.15 (m,
2H), 7.51 (br s, 1H), 8.15 (s, 1H). C NMR (CDCl3-d1)
d (ppm): 20.5,
26.0, 28.9, 51.8, 55.8, 104.6, 106.8, 109.1, 115.4, 118.3, 119.3, 120.3,
121.3, 121.5, 127.6, 128.9, 136.4, 145.3, 147.0, 147.9, 152.9.
3. (a) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52e67.
Compound 4c (as purple oil in 68% yield): H NMR (CDCl3-d1)
4. (a) Urano, Y.; Asanuma, D.; Hama, Y.; Koyama, Y.; Barrett, T.; kamiya, M.;
Nagano, T.; Watanabe, T.; Hasegawa, A.; Choyke, P. L.; Kobayashi, H. Nat. Med.
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d
(ppm): 1.16 (s, 3H),1.27 (s, 3H), 2.71 (s, 3H), 5.77 (d,1H, J¼10.3 Hz),
6.51 (d, 1H, J¼7.7 Hz), 6.79 (d, 1H, J¼10.3 Hz), 6.86 (dd, 1H, J¼7.2,
7.4 Hz), 7.05 (dd, 1H, J¼7.2, 7.4 Hz), 7.14e7.19 (m, 2H), 7.52 (br s, 1H),
7.71 (d, 1H, J¼2.4 Hz), 8.06 (s, 1H). C NMR (CDCl3-d1)
d (ppm): 20.4,
25.9, 28.9, 29.7, 52.1, 105.4, 106.9, 112.3, 119.6, 120.1, 121.3, 121.5,
127.8, 128.1, 128.3, 130.6, 136.1, 144.1, 147.6, 151.4. HRMS (MALDI-
TOF) for C20H19BrN2O2 calcd 401.0684 (MþHþ), found, 401.0669.
Compound 4d (as purple oil in 50% yield): H NMR (CDCl3-d1)
d
(ppm): 1.18 (s, 3H), 1.31 (s, 3H), 2.74 (s, 3H), 5.77 (d,1H, J¼10.2 Hz),
6.52 (d, 1H, J¼7.7 Hz), 6.84 (dt, 1H, J¼7.5, 0.8 Hz), 6.91 (d, 1H,
J¼10.2 Hz), 7.05 (dd, 1H, J¼7.2, 7.4 Hz), 7.16 (dd, 1H, J¼7.4, 7.6 Hz),
7.22e7.30 (m, 2H), 7.38 (t, 2H, J¼7.3 Hz), 7.53 (d, 2H, J¼7.1 Hz), 7.85
7. Wojtyk, J. T. C.; Wasey, A.; Xiao, N.-N.; Kazmaier, P. M.; Hoz, S.; Yu, C.; Lemieux,
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(d, 1H, J¼2.2 Hz), 8.20 (s, 1H). C NMR (DMSO-d6)
d (ppm): 20.0, 25.5,
28.6, 51.5, 79.1, 104.8, 106.9, 118.8, 119.2, 119.7, 120.2, 121.4, 122.9,
126.1, 126.4, 127.1, 127.5, 128.9, 129.2, 132.3, 136.1, 139.4, 141.7,
147.73, 147.6, 151.0. MALDI-TOF [MþH]þ calculated for C26H25N2O2:
10. Henary, M. M.; Fahrni, C. J. J. Phys. Chem. A 2002, 106, 5210e5220.