Fluorinated squaraine as near-IR label with improved properties for the labeling of oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2009.02.029

A new squaraine dye with fluorinated benzothiazole rings was synthesized. This new label possesses improved photophysical properties and chemical stability as compared to the corresponding non-fluorinated and the dicyanosquaraines. These squaraines were used for the labeling of a series of oligonucleotides with various sequences, lengths, and chemistries. The conjugates involving the fluorinated squaraine possess the best properties: emission wavelength >670 nm, high quantum yields (0.27-0.39).

Full text of DOI:10.1016/j.tetlet.2009.02.029

Tetrahedron Letters 50 (2009) 1897–1901
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fluorinated squaraine as near-IR label with improved properties for the
labeling of oligonucleotides
*
Brice-Loïc Renard, Yves Aubert, Ulysse Asseline
Centre de Biophysique Moléculaire, CNRS UPR 4301, Affiliated to the University of Orléans and to INSERM, Rue Charles Sadron, 45071 Orléans Cedex 02, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new squaraine dye with fluorinated benzothiazole rings was synthesized. This new label possesses
improved photophysical properties and chemical stability as compared to the corresponding non-fluori-
nated and the dicyanosquaraines. These squaraines were used for the labeling of a series of oligonucle-
otides with various sequences, lengths, and chemistries. The conjugates involving the fluorinated
squaraine possess the best properties: emission wavelength >670 nm, high quantum yields (0.27–0.39).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 9 January 2009
Revised 29 January 2009
Accepted 3 February 2009
Available online 8 February 2009
Keywords:
Fluorinated squaraine
Near-infrared label
Squaraine–oligonucleotide conjugates
1. Introduction
fluorescein3a and thiazole orange9 are improved as compared to
those of the unsubstituted ones, we have tested the influence of
The visualization of biomolecules in vivo can provide informa-
tion about their location, kinetics, and function. Among these
applications, the selective detection of specific DNA and RNA se-
quences can be achieved by using oligonucleotide probes comple-
mentary to the target sequence and containing a reporter group
that can be monitored using fluorescence spectroscopy.1 The
development of oligonucleotide-based therapeutic strategies can
also take advantage of the possibility to track the labeled oligonu-
cleotides during cell internalisation and cell trafficking toward
their targets. These applications require the use of labels that emit
in the near-infrared (NIR) range because beyond 600 nm the auto-
fluorescence of biological samples decreases.2 Numerous long-
wavelength labels belonging to rhodamine,3 BODIPY,4 oxazine,5
and cyanine dyes6 have been reported, and all have both associated
advantages and disadvantages depending on the intended applica-
tions. The synthesis and spectral characterization of other NIR la-
bels, derived from squaric acid, and their modification for
covalent attachment to biomolecules such as cholesterol, sugar
and proteins have also been reported.7,8 To date, despite suitable
spectroscopic properties, none of these compounds have been used
for the labeling of oligonucleotides. We report now the synthesis
and the photophysical properties of a series of five oligonucleo-
tides, with different sequences, lengths, and chemistries, labeled
with squaraine dyes based on benzothiazole. Since it has been pre-
viously reported that the photophysical properties of fluorinated
fluorination on the properties of the squaraine dyes and the corre-
sponding conjugates (Figs. 1 and 2).
2. Results and discussion
For the labeling of the oligonucleotides, we chose to use squa-
raines derived from benzothiazole (Scheme 1) and to proceed via
the post-synthetic coupling strategy. One benzothiazole ring was
methylated,10 and the second was alkylated with a diiodohexyl lin-
ker in order to react the squaraines with a thiophosphate group
0.2
0.15
0.1
0.05
0
250
350
450
550
650
750
Wavelength (nm)
Figure 1. UV–visible absorption spectra of 1 lM solutions of the conjugates 10SQFBt
(dotted lines), 12SQFBt (bold line), and 13SQFBt (plain line) in 10 mM cacodylate
buffer, pH 7, containing 100 mM NaCl recorded at 20 °C between k = 250 and
k = 800 nm.
* Corresponding author. Fax: +33 2 38 63 15 17.
E-mail address: ulysse.asseline@cnrs-orleans.fr (U. Asseline).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.02.029

1898
B.-L. Renard et al. / Tetrahedron Letters 50 (2009) 1897–1901
The synthesis of the squaraine dyes 1–3 was performed via a
multi-step procedure (Scheme 1). The dibutyl ester of the squaric
acid 5 was first reacted with the methylated benzothiazole deriv-
ative12 4a or with the corresponding fluorinated derivative 4b,
obtained by reaction of iodomethane excess with either benzothi-
azole or fluorobenzothiazole moieties, to give hemisquaraines 7a
and 7b. The latter were then reacted with the benzothiazole nu-
clei involving the iodohexyl linker attached to the nitrogen atom
of 8a and 8b, obtained by reaction of the benzothiazole and the
fluorobenzothiazole with 1,6-diiodohexane, to give the squaraines
1 and 2 in moderate yields. For the synthesis of squaraine 3
involving a dicyanomethylene group on the squaric acid, we chose
a similar strategy. First, the monobutyl ester of the dicyanomethyl
squaric acid 6 was obtained following a reported procedure.8a The
ester was reacted with the methylated benzothiazole 4a to give
hemisquaraine 7c. The dicyanomethylated squaraine 3 was then
obtained by reaction of 7c with the benzothiazole linker deriva-
tive 8a. All the compounds were characterized by NMR and
mass spectrometry analysis (See Refs. 13–15 and Supplementary
data).
Figure 2. Fluorescence emission spectra of 1 lM solutions of the conjugates 10SQFBt
(dotted lines), 12SQFBt (bold line), and 13SQFBt (plain line) in 10 mM cacodylate
buffer, pH 7, containing 100 mM NaCl recorded at 20 °C between k = 650 and
k = 750 nm.
S
N
X
I
In order to test the influence of the sequence, length, and chem-
istry of the oligonucleotide on the properties of the squaraine-oli-
gonucleotide conjugates, we used five different sequences (Table
1). The 50-thiophosphorylation was performed by our previously
published procedure.16 For the labeling step, different procedures
were tested, either in methanol solutions of crown ethers to solu-
bilize the oligonucleotides or in different mixtures of bicarbonate
buffer and dimethylformamide, in order to increase the yields in
conjugates. The coupling of squaraines 1 and 2 was successful with
the five oligonucleotides 9–13 (Scheme 2 and Table 1). The corre-
sponding conjugates 9SQBt–13SQBt and 9SQFBt–13SQFBt were ob-
tained in yields ranging from 3% to 24% after purification by
reversed-phase chromatography. The presence of the fluorine
atoms on the benzothiazole nuclei led to conjugates with slightly
increased retention times. Their masses were confirmed by mass
spectrometry analysis. The preparation of the conjugates of the
dicyanomethylated squaraine 3 failed despite the various condi-
tions used. This may be due to the propensity of the dye to form
aggregates and to its lack of stability under the reaction conditions
used.
X = H 4a (64%)
X = F 4b (83%)
O
NC CN
a
b
BuO
O
O
BuO
6
5
OBu
S
O
HNEt3
Y
Z
X = H, Y = O, Z = OBu 7a (40%)
X = F, Y = O, Z = OBu 7b (80%)
X = H, Y = C(CN)2 7c (50%)
O
N
X
Z =
O
HNEt3
S
N
X = H 8a (45%)
X = F 8b (45%)
(CH2)6I
c
X
I
(CH2)6I
Y
X
N
S
S
N
X
O
The dyes 1–3 were studied by absorption and fluorescence
spectroscopy. The presence of the fluorine atoms (compound 2) in-
duced a red shift of 6 nm in the absorption spectra (k = 662 vs
656 nm for 1) in EtOH and (k = 675 vs 669 nm) in chloroform.
The presence of the dicyanomethyl group on the squaric acid in-
duced a more pronounced red shift (+25 nm) for compound 3.
X = H, Y = O
X = F, Y = O
1
= SQBt(CH2)6I (10%)
= SQFBt(CH2)6I (23%)
= SQDCBt(CH2)6I (15%)
2
3
X = H, Y = C(CN)2
Scheme 1. Synthesis of the squaraine dyes 1–3. Reagents and conditions: (a) EtOH,
TEA, 65 °C, 90 min; (b) EtOH, TEA, 65 °C, 60 min; (c) butanol, toluene, 120 °C, 10 h,
or 100 °C, 8 h for X = H, Y = C(CN)2.
The e
values were high (P200,000 Mꢀ1 cmꢀ1) as were the quantum
yields (ꢁ0. 44 for 1 and 2 and 0.35 for 3 in CHCl3).
incorporated at the 50-end of the oligonucleotides to form a phos-
phothiolodiester linkage (Scheme 2).11
When the solutions were stored at 4 °C, no change in emission
intensity was observed over the course of 15 h. At 20 °C, a 3%
O
5'
3'
OH
ON
SQBt-(CH ) -
S
P
O
2 6
1
O
P
O
NH4
5'
3'
OH
9SQBt 10SQBt
12SQBt 13SQBt
11SQBt
ON
S
O
a
O
2 NH4
O
5'
3'
OH
2
SQFBt-(CH ) -
ON
S
P
O
2 6
9-13
O
NH4
9SQFBt 10SQFBt
11SQFBt 12SQFBt 13SQFBt
Scheme 2. Synthesis of the conjugates labeled with 1 and 2. Reagents and conditions: (a) 2.5% aqueous bicarbonate buffer, pH 9 (or pH 7 for 2), (0.15 mL). The mixture was
stirred for 4–10 h at rt.

B.-L. Renard et al. / Tetrahedron Letters 50 (2009) 1897–1901
1899
Table 1
Characterizations for conjugates
ONs
Sequences
Rt (min) HPLC
Mass analysis
Yield (%)
0
0
9
10
11
12
5 p(S)TTTTCTTTTC3
11 min 37 s
9 min 41 s
3044.00
3044.06
3122.85
4221.70
4318.94
6438.40
3519.03
4695.55
4695.55
4791.56
6912.38
3554.90
3629.30
4729.64
4827.99
6946.70
70
65
50
58
66
6
5
6
8
7
24
4
3
5
8
0
0
5 p(S)ATTTGGAACC3
3123.08
4221.77
4318.87
6438.97
3519.63
4694.39
4694.39
4791.48
6910.59
3553.62
3630.68
4730.37
4827.46
6946.57
0
0
5 p(S)TTCTCCCCCGCTTA3
12 min 40 s
10 min 43 s
12 min 50 s
27 min 06 s
27 min 10 s
26 min 22 s
26 min 33 s
27 min 31 s
28 min 03 s
27 min 34 s
26 min 59 s
27 min 58 s
27 min 58 s
0
0
5 p(S)CCGCTTAATACTGA3
30
0
13
5 pðSÞðU-20OMeÞ20
0
0
9SQBt
10SQBt
11SQBt
12SQBt
13SQBt
9SQFBt
10SQFBt
11SQFBt
12SQFBt
13SQFBt
SQBt-(CH2)6-5 SpTTTTCTTTTC3
0
0
SQBt-(CH2)6-5 SpATTTGGAACC3
0
0
SQBt-(CH2)6-5 SpTTCTCCCCCGCTTA3
0
0
SQBt-(CH2)6-5 SpCCGCTTAATACTGA3
30
0
SQBt-ðCH2Þ6-5 SpðU-20OMeÞ20
0
0
SQFBt-(CH2)6-5 SpTTTTCTTTTC3
0
0
SQFBt-(CH2)6-5 SpATTTGGAACC3
0
0
SQFBt-(CH2)6-5 SpTTCTCCCCCGCTTA3
0
0
SQFBt-(CH2)6-5 SpCCGCTTAATACTGA3
30
0
SQFBt-ðCH2Þ6-5 SpðU-20OMeÞ20
ONs: 50-thiophosphorylated oligonucleotides and oligonucleotide-squaraine conjugates. Retention times obtained by reversed-phase chromatography analyses for the 50-
thiophosphorylated ONs 9–13 and the corresponding conjugates performed on a Lichrospher RP 18 (5
lm) column (125 mm ꢂ 4 mm) from Merck using a linear gradient of
CH3CN (5–38.5% over 45 min) in 0.1 M aqueous ammonium acetate, pH 7, with a flow rate of 1 mL/min. Mass analysis data for the 50-thiophosphorylated ONs 9–13 and the
corresponding conjugates. Yields for the 5-thiophosphorylated oligonucleotides and conjugates.
decrease was observed for 1 and only 2% for 2 over the same period
of time. At 37 °C, no real change was observed over 4 h for 1 and 2.
The absorption spectra of conjugates 9–13 were recorded be-
tween k = 250 and k = 800 nm. In all cases, the spectra contain an
absorption band in the UV range corresponding to the absorbance
of the oligonucleotide and the squaraine, while the absorption
band in the visible range corresponds to that of the squaraine.
The kmax values of both the UV and visible absorption bands are gi-
ven in Table 2. The intensity ratios of the UV–visible bands are also
different depending on the squaraine considered (Table 2). A com-
parison of the two series of conjugates showed that for every squa-
raine, the kmax visible was slightly different depending on the
sequence, suggesting interactions between the squaraines and
However, different fluorescence intensities were observed. The
presence of the fluorine atoms induced intensity increases by
about 50%. All the conjugates exhibited high quantum yields (Table
2) with the highest values (0.27–0.39) for those involving the
fluorinated squaraine 2.
The photostability of the squaraine linker derivatives 1–2 and of
oligonucleotide-squaraine conjugates 9SQBt, 9SQFBt, 10SQBt, and
10SQFBt was investigated and compared to those of the correspond-
18
ing conjugates of the well-known dyes thiazole orange 9TO and
Indocyanine Green analogue6i,19 9Cy7mh involving the dyes at-
tached via the same linker (See Supplementary data). For the
photostability measurements, 1
chloroform or ethanol for compounds 1–2 and in a 10 mM cacodyl-
ate buffer, pH 7, containing 100 mM NaCl for the conjugates 9SQBt
lM solutions were prepared in
the oligonucleotides. The molar extinction coefficient values (
e
)
,
for conjugates 9SQBt, and 9SQFBt were determined by titration of
9SQFBt, 10SQBt and 10SQFBt, 9TO and 9Cy7mh. Ten successive emission
spectra were recorded at room temperature with the same solu-
tions. No changes in the emission were observed for 2 and only
5% for 1 in ethanol. The same experiment performed with the con-
jugates indicated a 10% emission decrease for the conjugates
involving the fluorinated cyanine versus 25% for the non-fluori-
nated ones. Under the same conditions, the emission of conjugates
9TO and 9Cy7mh remained unchanged.
0
their solutions with the complementary sequence 5 GGGAAA
0
AGAAAATTT3 at 4 °C. For the conjugates of 10–13 the
e
values at
260 nm were approximated as the sum of the
e values of the
corresponding oligonucleotides, determined using the nearest
neighbor model,17 and of the squaraines deducted from those of
conjugates 9SQBt and 9SQFBt
.
A comparison of the emission spectra of the two series of con-
jugates clearly indicated similar emission shapes with kmax emis-
sion around 670 nm depending on the oligonucleotide sequences.
Solutions of the conjugates 9SQBt and 9SQFBt in a 10 mM cacodyl-
ate buffer, pH 7, containing 100 mM NaCl were stored at different
Table 2
Absorption and fluorescence data for conjugates
Conjugates
Absorption
kVISmax (nm)
Fluorescence
kUVmax (nm)
e
(Mꢀ1 cmꢀ1
)
e
(Mꢀ1 cmꢀ1
)
Conjugates
Conjugates
kex (nm)
kem (nm)
If
/
9SQBt
268
260
265
262
259
266
260
266
262
260
87,100
654
656
659
657
649
661
662
666
661
655
114,300
128,400
102,200
108,600
100,600
122,000
126,600
119,300
114,300
102,000
654
656
659
657
649
661
662
666
661
665
668
670
671
671
666
673
676
678
675
671
111.48
137.05
103.36
100.00
61.83
165.34
177.56
176.33
160.00
110.27
0.24 (0.16 EtOH)
0.26
0.24
0.23
0.17
0.32 (0.20 EtOH)
0.37
0.39
0.35
0.27
10SQBt
11SQBt
12SQBt
13SQBt
9SQFBt
10SQFBt
11SQFBt
12SQFBt
13SQFBt
105,100
122,700
140,600
169,600
85,500
103,500
121,100
139,000
168,000
Absorption and emission spectra were recorded in a 10 mM sodium phosphate buffer, pH 7, containing 140 mM KCl and 5 mM MgCl2 at 20 °C. Concentrations were 1
conjugates. The values in parenthesis correspond to the / of the squaraines 1 and 2 determined in ethanol.
lM in
Molar absorption coefficients (e) were determined experimentally for the conjugates 9SQBt and 9SQFBt. The e values at k = 260 nm for the other conjugates were the sum of the
e
values for the oligonucleotides and the squaraines.

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undetectable after 4 h at 20 °C or 1 h at 37 °C, that of 9SQFBt was
70% of its initial value after 4 h at 20 °C and was 75% after 1 h at
37 °C. The fluorescence decrease observed at 20 °C was similar to
that obtained with the conjugate 9Cy7mh, while that of 9TO was
unchanged.
5. Jose, J.; Burgess, K. Tetrahedron 2006, 62, 11021–11037.
6. (a) Hammer, R. P.; Owens, C. V.; Hwang, S.-H.; Sayes, C. M.; Soper, S. A.
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13. 4-[N-(6-iodohexyl)-1,3-benzothiazoliumyl-2-methylene]-2-(N-methyl-2,3-dihydro-
1,3-benzothiazol-2-ylidenemethyl)-3-oxo-1-cyclobutene-1-olate 1: Compound 7a
(380 mg, 1 equiv, 1.20 mmol) was added to a solution of 8a (587 mg, 1 equiv,
1.20 mmol) in a butanol/toluene (1:1, v/v) mixture (15 mL). The mixture was
stirred at 120 °C for 10 h. After removal of the solvent by evaporation, the
residue was purified on a silica gel column using a MeOH gradient (0–5%) in
CH2Cl2, then on preparative silica gel plates using a CH2Cl2/MeOH mixture (96:4,
v/v) as eluent to give a dark blue solid 42 mg. Yield: 12%. TLC: Rf (CH2Cl2/MeOH
8/2) = 0.45. 1H NMR (500 MHz, CDCl3, TMS): d (ppm) = 7.61 (t, 2H, J = 7.8 Hz,
HAr), 7.47 (m, 2H, HAr), 7.32 (m, 4H, HAr), 6.39 (s, 2H, = CH), 4.18 (m, 2H, +N–
CH2–), 3.75 (s, 3H, N–CH3), 3.24 (t, J = 6.9 Hz, 2H, –CH2I), 1.88 (m, 4H, 2 CH2–),
1.56 (m, 4H, 2 CH2–). 13C NMR (126 MHz, CDCl3, TMS): d (ppm) = 6.8, 26.1, 27.4,
30.4, 33.0, 33.4, 46.2, 85.6, 111.4, 111.5, 122.3, 122.4, 124.1, 124.2, 127.2, 127.3,
128.7, 128.9, 141.2, 141.7, 159.9, 160.1. Maldi-Tof-MS: m/z, C27H25O2N2S2I calcd
600.55, found 600 (M+).
The influence of the pH was also tested. The emission spectra of
solutions of conjugates 9SQBt, 9SQFBt, 10SQBt, and 10SQFBt, were re-
corded in aqueous buffer with pH ranging from 5 to 8. The results
indicated nearly equivalent values at any pH for the conjugates
involving the fluorinated squaraine, while the conjugate derived
from the non-fluorinated squaraine was not stable at pH below 7.
The presence of the squaraines induced a weak stability in-
crease of the complexes formed by the conjugates 9SQBt and 9SQFBt
0
0
with their single-stranded 5 GGGAAAAGAAAATTT3 and double-
0
0
0
stranded 5 GCCACTTTTTAAAAGAAAAGGGG3 /3 CGGTGAAAAATTT
0
TCTTTTCCCC5 DNA targets. Upon hybridization at 20 °C, the fluo-
rescence emission of the 9SQBt and 9SQFBt decreased by 28% and
9% in the presence of the single-stranded target and by 8% and
42% in the presence of the double-stranded target.
3. Conclusion
We have reported the synthesis of three squaraine dyes based
on benzothiazole moiety with wavelength emissions beyond
670 nm. Two of them have been successfully used for the labeling
of a series of five oligonucleotides with different sequences,
lengths, and chemistries. The oligonucleotides labeled with the
squaraines exhibited high fluorescent emissions, about 60 and
30-fold those of the corresponding oligonucleotides labeled with
the well-known dyes thiazole orange and Indocyanine Green ana-
logue. As previously reported for thiazole orange, the fluorination
of the benzothiazole heterocycle of the squaraine led to an
improvement in photostability. This photostability increase was
also observed with the labeled oligonucleotides. The presence of
the fluorine atom on the label induced a strong chemical stability
increase, between pH 5 and 8, that reached that of the correspond-
ing thiazole orange labeled oligonucleotide. These properties make
the fluorinated squaraine dyes very promising tools for the labeling
of oligonucleotides.
Acknowledgments
14. 4-[5-Fluoro-N-(6-iodohexyl)-1,3-benzothiazoliumyl-2-methylene]-2-(5-fluoro-
N-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidenemethyl)-3-oxo-1- cyclobutene-
We thank the Region Centre for a doctoral fellowship to B.-L.R.,
the «Plateforme de Spectrometrie de masse» of the «Centre de Bio-
physique Moléculaire» for analysis facilities, G. Gabant for running
the mass spectrometers, and H. Meudal for recording the NMR
spectra.
1-olate 2: Compound 7b (440 mg, 1 equiv, 1.32 mmol) was added to
a
solution of 8b (668 mg, 1 equiv, 1.32 mmol) in a butanol/toluene (1:1, v/v)
mixture (15 mL). The mixture was stirred at 120 °C for 10 h. After removal
of the solvent by evaporation, the residue was purified on
a silica gel
column using a MeOH gradient (0–3%) in CH2Cl2, then on preparative silica
gel plates using a CH2Cl2/MeOH mixture (96:4, v/v) as eluent to give a dark
blue solid 91 mg. Yield: 23%. TLC: Rf (CH2Cl2/MeOH 8/2) = 0.45. 1H NMR
(500 MHz, CDCl3, TMS): d (ppm) = 7.47 (m, 2H, HAr), 6.96 (m, 2H, HAr), 6.89
(m, 2H, HAr), 5.91 (s, 2H, @CH), 4.06 (t, 2H, J = 7.8 Hz, +N–CH2–), 3.64 (s, 3H,
N–CH3), 3.23 (t, J = 6.9 Hz, 2H, –CH2I), 1.86 (m, 4H, 2 CH2–), 1.52 (m, 4H, 2
CH2–). 13C NMR (126 MHz, CDCl3, TMS): d (ppm) = 6.7, 26.1, 27.3, 30.4, 33.2,
33.3, 46.6, 86.5 (d, J = 14.7 Hz), 99.4 (d, JC–F = 28.1 Hz), 99.6 (JC–F = 28.1 Hz),
111.7 (t, JC–F = 23.1 Hz), 123.2 (t, JC–F = 10.5 Hz), 161.0, 162.7 (d,
JC–F = 245.7 Hz). Maldi-Tof-MS: m/z, C27H23O2N2S2F2I calcd 636.52, found
637.27 (M+).
Supplementary data
The Supplementary data section includes detailed experimental
conditions and characterization data for compounds 4a, 4b, 6, 7a,
7b, 7c, 8a, 8b and Cy7mh and 9Cy7mh. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.02.029.
15. 3-Dicyanomethylene-4-[N-(6-iodohexyl)-1,3-benzothiazoliumyl-2-methylene]-2-
(N-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidenemethyl)-1-cyclobutene-1-olate
3: Compound 7c (270 mg, 1 equiv, 0.66 mmol) was added to a solution of 8a
(362 mg, 1.2 equiv, 0.802 mmol) in EtOH (10 mL), and the mixture was heated
to 85 °C for 8 h under stirring. After removal of the solvent by evaporation, the
residue was purified on a silica gel column using a MeOH gradient (0–5%) in
CH2Cl2, then on preparative silica gel plates using a CH2Cl2/MeOH mixture
(98:2, v/v) as eluent to give a turquoise blue solid. Yield: 15%. 1H NMR
(500 MHz, CDCl3, TMS): d (ppm) = 7.57 (t, 2H, J = 7.5 Hz, HAr), 7.41 (d t, 2H,
J = 7.5 Hz, J = 8 Hz, HAr), 7.24 (q, 2H, J = 7 Hz, HAr), 7.17 (m, 2H, HAr), 5.92 (s, 2H,
@CH), 4.12 (t, 2H, J = 7.5 Hz, –+N–CH2–), 3.68 (s, 3H, N–CH3), 3.23 (t, J = 7 Hz,
2H, –CH2I), 1.88 (m, 4H, 2 CH2–), 1.52 (m, 4H, 2 CH2–). 13C NMR (126 MHz,
CDCl3, TMS): d (ppm) = 7.1, 26.0, 27.6, 29.9, 30.3, 33.3, 46.9, 87.3, 112.0, 112.1,
References and notes
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1901
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