Thiazole Orange derivatives: Synthesis, fluorescence properties, and labeling cancer cells

Article abstract of DOI:10.1016/j.bmc.2008.11.083

A series of Thiazole Orange (TO) derivatives were synthesized and modified by introducing different substitutional groups on benzothiazole and 4-methylquinoline All the TO derivatives were confirmed by ¹HNMR and MS. TO derivative bearing NH

Full text of DOI:10.1016/j.bmc.2008.11.083

Bioorganic & Medicinal Chemistry 17 (2009) 585–591
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiazole Orange derivatives: Synthesis, fluorescence properties,
and labeling cancer cells
*
Xuening Fei , Yingchun Gu, Ying Ban, Zhijun Liu, Baolian Zhang
Department of Material Science and Engineering, Tianjin Institute of Urban Construction, Tianjin 300384, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Thiazole Orange (TO) derivatives were synthesized and modified by introducing different sub-
stitutional groups on benzothiazole and 4-methylquinoline All the TO derivatives were confirmed by
1HNMR and MS. TO derivative bearing NH2– was modified by folic acid and used to label breast cancer
cells. The phenomenon of fluorescence enhancement was shown by the fluorescence spectrums of TO
derivatives and micrographs of the labeled breast cancer cells. It offered a new try in the aspect of label-
ing cells by the embedded dyes.
Received 24 September 2008
Revised 26 November 2008
Accepted 27 November 2008
Available online 11 December 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Thiazole Orange
Modification
Fluorescence
Label
Breast cancer cells
1. Introduction
lated chitosanthiocyanate to get targeted image for hepatocytes.
It was found that optical images were mainly concentrated in
The 4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methyli-
dene]-quinoliniumiodide fluorophore, better known as cyanine
dye Thiazole Orange (TO), possesses many distinctive and desirable
properties: it has very low intrinsic fluorescence, chemical stabili-
ties, and a high molar absorption coefficient.1 The dye is virtually
non-fluorescent when not combined with nucleic acids, but shows
fluorescence enhancement over 1000-fold when inserted into DNA,
especially into double-helix of DNA, and over 3000-fold when in-
serted into RNA.2–8 This fluorescence character can be used to im-
prove the detection sensitivity, which reduces background
interference that is difficult to remove from intracellular environ-
ments or tissues.9,10 Moreover, the dye has higher affinity to tumor
than to the normal cells, which will be widely used in early-stage
labeling of cancer cells.11
Recently, considerable efforts have been devoted to the mod-
ification of TO derivatives because of the important roles that
they play in various chemical and biological processes. Chitosan
oligosaccharide (CTS), a typical bioactive polysaccharide,12,13 is
easy to be modified selectively by appropriate biological mole-
cules based on special object, which provides a new method
for traditional detection in biomedicine investigation. As a non-
viral vector, galactosylated chitosan showed good targeting effect
when used in gene therapy for hepatocellular carcinoma.14 In
2005, Kim et al.15 used fluorescent dyes modified by galactosy-
hepatocytes at 10, 60, 120 min after injection respectively. CTS
acts as a bridge between targeted cancer cells and fluorescent
dyes, which can enhance the cellular compatibility with dyes
and reduce toxicity.
A variety of different targeting strategies are currently under
investigation to enhance the specificity of labeling tumor. Folic
acid has shown potential as a targeting device because the folate
receptor is highly overexpressed in a number of human tumors
including breast cancer, ovarian cancer, cervical cancer, colorectal
cancer, nasopharyngeal cancer, lung cancer, whereas in normal tis-
sue, its expression is significantly lower, which may be ideally sui-
ted for the study of diagnosis and therapy of cancers.16–25
Kennedy et al.26 used cyanine dyes modified by folic acid to
study the optical imaging of tumor cells. It was found that tumor
cells with folate receptor showed bright signs but normal cells
hardly. This method can distinguish tumor cells from normal cells.
In the present study, we have prepared a series of designed mol-
ecules of which different substitutional groups have been intro-
duced to benzothiazole and 4-methylquinoline. TO and its
derivatives were synthesized using the reaction scheme as shown
in Scheme 1. These TO derivatives share a common core structure.
TO derivatives with an amine residue can be modified by folic acid
easily for the study of identifying cancer cells as targets that have
an extra folacin acceptor on the cellular surface. By connecting CTS
to TO derivative with carboxyl residue (TO–COOH) to gain TO
derivative (TO–COOH–CTS), a new labeling strategy also has been
developed.
* Corresponding author. Tel.: +86 22 23085033; fax: +86 22 23085032.
E-mail address: xueningfei@126.com (X. Fei).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.083

586
X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
K2CO3
CH3OTs
S
N
S
N
+
SH
CH2Br
R
S CH2
R
CH3OH
reaction (I)
reaction (II)
1a-1e
2a-2e
1a: R=H,
1b: R=6-CH3,
1c: R=5-Cl,
1d: R=5-NO2
1e: R=6-NO2
2a: R=H,
2b: R=6-CH3,
2c: R=5-Cl,
2d: R=5-NO2
2e: R=6-NO2
S
S CH2
R
N
OTs
3a-3e
S
3a: R=H,
3b: R=6-CH3,
3c: R=5-Cl,
3d: R=5-NO2
3e: R=6-NO2
Et3N
X
R
C
H
OTs
N
N
5a-5k
X
N
8h
TO{5a-5k} a: R=H, X=Br; b: R=H, X=NH2;
c: R=H, X=COOH; d: R=6-CH3, X=Br;
e: R=6-CH3, X=COOH; f: R=5-Cl, X=Br;
g: R=5-Cl, X=COOH; h: R=5-NO2, X=Br;
i: R=5-NO2, X=COOH; j: R=6-NO2, X=Br;
k: R=6-NO2, X=COOH
+
BrCH2CH2X
Br
N
4a-c
4a: X=CH2Br
4b: X=CH2NH2
4c: X=COOH
Scheme 1. Synthetic routes of the TO derivates.
2. Results and discussion
2.1. Synthesis
Table 2
The effect of reaction time on the synthesis of 2a
Entry
Time (h)
Yield (%)
As a traditional alkylating agent, iodomethane is expensive and
of high toxicity, dimethyl sulfate is also virulent. Benzyl bromide
was used as the alkylating agent to decrease the cost, toxicity,
and reduce the damage to our environment in this paper. The influ-
ence factors of the alkylation were investigated, such as solvent,
temperature, and reaction time. A series of solvents used in the
experiment was studied separately and the results were shown
in Table 1. It was found that the higher data of dielectric constant
and lower data of dipole moment were useful factors for the
increasing yield from entries 1 to 5. Based on the optimum reaction
condition from Table 1, entry 5, the suitable reaction time was also
studied, which was shown in Table 2. The desirable result was
displayed in entry 2 (1 h, 93%). The yield hardly increased by pro-
longing reaction time. Moreover, the conditions of quaterization of
2-benzylmercaptobenzothiazole were also optimized. The appro-
priate reaction time, reaction temperature and solvent were con-
cluded from data shown in Table 3–5, and the highest yield
(78%) was obtained in refluxing xylene for 24 h. Solvent polarity
is one of the major factor affecting the reaction process. The result
revealed that the reaction temperature was the more important
factor when the solvent data of the dielectric moment was similar
to the other ones.
1
2
3
0.5
1
2
83
93
93
spectrum of TO bearing –NH2 (TO–NH2) and its derivates were
shown in Figure 1 and TO–COOH derivates in Figure 2. From the
IR curves of TO–NH2 and TO–NH2 derivative (folate–TO–NH2)
modified by N-hydroxysuccinimide eater of folic acid (NHS–folate),
obvious differences of the absorbing peaks position between them
can be found.
As shown in Figure 1, the spectrum peaks at 1695 cmꢀ1
,
1297 cmꢀ1, 1188 cmꢀ1, 839 cmꢀ1, 820 cmꢀ1, 450 cmꢀ1 were new
ones in folate–TO–NH2 compared to the spectroscopy curve of
TO–NH2 but they were found in the curve of folic acid at
1694 cmꢀ1
,
1299 cmꢀ1
,
1192 cmꢀ1
,
839 cmꢀ1
,
819 cmꢀ1
,
445 cmꢀ1, respectively. Thus TO–NH2 is modified by NHS–folate
to gain folate–TO–NH2. From Figure 2, it was found that the curves
of folate–TO–COOH–CTS and TO–COOH–CTS were very similar, but
new spectrum peaks appeared at 1608 cmꢀ1, 952 cmꢀ1, 848 cmꢀ1
,
618 cmꢀ1, whose relevant peaks could be found in the curve of folic
acid at 1606 cmꢀ1, 973 cmꢀ1, 839 cmꢀ1, 593 cmꢀ1, respectively. It
is shown that folic acid is combined with TO–COOH–CTS to afford
folate–TO–COOH–CTS.
2.2. Chemical structure identification of modified TO derivates
According to DTA thermograms results, a endothermic peak at
124 °C and a exothermic peak at 195 °C was on the DTA curve of
folic acid, while there was a exothermic peak at 181 °C on the
DTA curve of TO–NH2, but a exothermic peak at 282 °C on that of
The structures of modified TO derivates were determined by IR
spectrum and differential thermal analysis (DTA) spectrum. The IR
Table 1
The effect of solvent categories on the synthesis of 2a
Entry
Solvent
Dielectric constant27 (25 °C)
Dipole moment27 (D)
Temp. (°C)
Time (h)
Yield (%)
1
2
3
4
5
6
7
CH3COCH3
CHCl3
PhCH3
20.70
4.81
2.38
2.86
1.15
1.33
1.89
1.73
1.73
1.70
Reflux/56
Reflux/61
Reflux/111
Reflux/77
Reflux/78
rt
4
1
1
1
1
1
1
62
71
70
82
88
23
93
EtOAc
6.02
C2H5OH
C2H5OH
CH3OH
24.55
24.55
32.70
rt

X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
587
Table 3
The effect of solvent categories on the synthesis of 3a
Entry
Solvent
Dielectric constant27 (25 °C)
Dipole moment27 (D)
Time (h)
Temp. (°C)
Yield (%)
1
2
3
4
5
6
PhCH3
2.38
—
20.70
6.02
24.55
4.81
1.33
—
2.86
1.89
1.73
1.15
24
24
24
24
24
24
Reflux/111
Reflux/140
Reflux/56
Reflux/77
Reflux/78
Reflux/61
32
78
0
0
0
Ph(CH3)2
CH3COCH3
EtOAc
CH3CH2OH
CHCl3
2
Table 4
folate–TO–NH2. It is further illustrated that folate–TO–NH2 is
bonded by folic acid and TO–NH2. To summarize, folate–TO–NH2
was proved to be a new compound reacted by NHS–folate and
TO–NH2 with infrared spectroscopy and the DTA thermograms.
There was only a slow weightloss on the DTA curve of TO–
COOH–CTS and there was a endothermic peak at 124 °C and a exo-
thermic peak at 195 °C on the DTA curve of folic acid with no
weightloss curve on it, but there were two exothermic peaks at
262 °C and 239 °C on the DTA curve of folate- TO–COOH–CTS with
a slow weightloss. As shown above, folate–TO–COOH–CTS is
proved to be a new compound bonded by folic acid and TO–COOH
and not a simple mixture of them by infrared spectroscopy and the
DTA thermograms.
The effect of reaction temperature on the synthesis of 3a
Entry
Temp. (°C)
Time (h)
Yield (%)
1
2
3
80
111
140
24
24
24
0
32
78
Table 5
The effect of reaction time on the synthesis of 3a
Entry
Time (h)
Yield (%)
1
2
3
24
36
48
77
78
78
2.3. Fluorescence properties
The fluorescence spectrums of TO derivatives were measured
and the results were shown in Figure 3. It was shown that the val-
ues of maximal fluorescence emission (kex) shift to a certain extent
when TO was modified by different substitutional group. TO deriv-
atives containing the nitro group had a higher data of kex; and
intensity. The special characteristics of their fluorescence didn’t
change obviously although the nitro group was at different posi-
tion on the aryl ring.
The fluorescence spectrums of TO–COOH and TO–COOH–CTS
were measured for comparing the differences of relevant fluores-
cence. The kex of TO–COOH–CTS was close to that of TO–COOH
with greatly increased fluorescence intensity and the phenomenon
of enhancement of fluorescence was shown obviously in Figure 4.
Based on this result, it may be possible to design and prepare a
kind of fluorescence probe of Thiazole Orange with high sensitivity,
good biological intermiscibility, and excellent water solubility. The
interaction relationship between TO–COOH and CTS are being
studied in our group.
folic acid
TO-NH2
folate-TO-NH2
2000
1500
1000
Wavenumbers (cm-1
500
)
The fluorescence spectrums of TO–NH2 and TO–COOH marked
by BSA were also measured and the results were shown in Figure
5. The kex of TO–COOH appeared a red shift with decreased inten-
Figure 1. IR spectrums of TO–NH2 derivatives.
folic acid
folate-TO-COOH-CTS
TO-COOH-CTS
1800
1600
1400
1200
1000
800
600
Wavenumbers (cm-1
)
Figure 2. IR spectrums of TO–COOH derivatives.
Figure 3. Fluorescence spectrums of TO derivatives.

588
X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
Figure 6. Fluorescence spectrums of folate–TO–NH2 and derivative marked by BSA.
Figure 4. Fluorescence spectrums of TO–COOH and TO–COOH–CTS.
sity compared to that of TO–NH2 and the intensity both increased
after marked with BSA. We also studied the fluorescence spec-
trums of folate–TO–NH2 and folate–TO–NH2–BSA shown in Figure
6. It revealed that the kex of folate–TO–NH2–BSA was close to that
of folate–TO–NH2 with increased fluorescence intensity.
2.4. Label of breast cancer cells
Embedded fluorescence dyes can combine with nucleic acid and
show fluorescence, such as TO, propidium iodide (PI). It is reported
that PI can combine with double stranded DNA but cannot pass
through the cell membrane.28 In this paper, we observed an inter-
esting phenomenon that TO and its derivatives can penetrate
through the cell membrane into the nucleus and show strong fluo-
rescence. TO–COOH and TO–COOH–CTS were used to study in
labeling breast cancer cells. The photomicrographs of breast cancer
cells incubated at 37 °C under the invert microscope were shown
in Figures 7 and 8, which offered a new try in the aspect of labeling
cells by the embedded dyes. Experimental results demonstrated
that green fluorescence was observed in cell nucleus but not in
the cytoplasm of the cells labeled by TO–COOH. However, in the
photos of cells labeled by TO–COOH–CTS, both of cell nucleus
and cytoplasm showed strong green fluorescence. Also, the cells la-
beled by TO–COOH–CTS showed stronger fluorescence than the
cells labeled by TO–COOH, which exhibited noticeable enhance-
ment of fluorescence. The result of cancer cells labeled by folate–
TO–COOH–CTS was shown in Figure 9. In the photo green fluores-
cence was observed in cell nucleus but not in the cytoplasm, which
appeared a similar case labeled by TO–COOH.
Figure 7. Micrographs of breast cancer cells labeled by TO–COOH.
Figure 8. Micrographs of breast cancer cells labeled by TO–COOH–CTS.
TO–NH2 and folate–TO–NH2 were also used in the labeling cells
and the results were shown in Figures 10 and 11. The strong bright
fluorescence signals were seen in the distribution of the cell nu-
cleus and not in the cell membrane of breast cancer cells labeled
by TO–NH2 and folate–TO–NH2. It is observed that TO–NH2 can
penetrate through the cell membrane and exist stably in the nucle-
ar with no toxicity.
Over repeated experiment, the labeled samples showed very lit-
tle photobleaching, far less than with conventional dye molecules.
So the structures of TO and its derivatives are not degraded and the
dyes could penetrate through the cell membrane into the nucleus
with no toxicity and show fluorescence and sense information.
Figure 5. Fluorescence spectrums of TO derivatives marked by BSA.

X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
589
4. Experimental
4.1. General experimental conditions
Fluorescence spectra were scanned on a fluorescence analysis
instrument, Cary Eclipse, American. Mass spectral analyses were ob-
tained using an electrospray ionization (ESI) mass spectrometer.
Melting points were taken on a Yanaco apparatus and are uncor-
rected. 1H NMR spectra were recorded on a Bruker AC-P300
(300 MHz) spectrometer. Chemical shifts are reported in parts per
million (ppm) downfield from TMS (tetramethylsilane), using
DMSO-d6 as a solvent. DTA thermograms were recorded on LCT-2
differential thermal balance (China). All the reagents are analytically
pure.
4.2. General procedure for the preparation of 2-
benzylmercaptobenzothiazole derivatives 2a–2e
Figure 9. Micrographs of breast cancer cells labeled by folate–TO–COOH–CTS.
2-Mercaptobenzothiazole derivatives 1 were prepared accord-
ing to traditional method29 which served as our starting materials
for further synthetic studies. 2-mercaptobenzothiazole derivatives
(8.97 mmol) were added to anhydrous methanol (30 mL) and stir-
red to dissolve, then K2CO3 (2.00 g , 11.30 mmol) and benzyl bro-
mide (2.00 mL, 10.00 mmol) were added to the mixture and
stirred at room temperature. TLC method followed the track of
reaction until the reaction completed.
Reaction mixture was filtered hot, and the filtrate was dried in
vacuo. The residue was separated on a flash chromatography col-
umn (silica gel, solvent ethyl acetate:hexane 50:1(v/v)) to afford
compounds 2. The results are shown in Table 6.
4.3. General procedure for the preparation of 2-
benzylmercapto-N-methylbenzothiazole derivatives 3a–3e
2-Benzylmercapto-N-methylbenzothiazole derivatives were per-
formed by refluxing 2-benzylmercaptobenzothiazole derivatives 2
(3.50 mmol) and methyl p-toluenesulfonate (1.30 g, 6.98 mmol) in
xylene (20 mL), followed by TLC method until the reaction com-
pleted. After dumping the upper solution of the mixture, the resi-
due was separated by column chromatography via silica gel with
methanol to yield compounds 3. The data are shown in Table 7.
Figure 10. Micrographs of breast cancer cells labeled by TO–NH2.
4.4. General procedure for the preparation of TO derivates 5a–5k
TO and its derivates were synthesized by mixing compounds 3
(2.00 mmol) and compounds 4 (2.72 mmol)30 in 30 mL ethanol. To
this solution, triethylamine (Et3N, 0.80 mL) was added causing the
reaction mixture to immediately turn a salmon pink color. After stir-
ringfor 60 min, theproductswere precipitated byaddition of diethyl
ether, filtrated, washed and dried to give salmon pink solid 5.
4.5. TO derivative (5a)
Yield: 97%, mp: 202–204 °C. 1H NMR (300 MHz, DMSO-d6): d
2.42 (t, J = 6.75 Hz, 2H), 3.66 (t, J = 6.30 Hz, 2H), 4.03 (s, 3H), 4.69
(t, J = 6.75 Hz, 2H), 6.94 (s, 1H), 7.36–7.46 (m, 2H), 7.62 (t,
Figure 11. Micrographs of breast cancer cells labeled by folate–TO–NH2.
3. Conclusions
In conclusion, eleven TO derivatives were synthesized in opti-
mal conditions and confirmed by 1HNMR and MS. The fluorescence
spectrums of TO derivatives were studied preliminarily and the
phenomenon of the enhancement of fluorescence was depicted.
Breast cancer cells labeled by TO derivative bearing –COOH or –
NH2 and their derivatives were also studied preliminarily, which
offered a new try in the aspect of labeling cells by the embedded
dyes. The multiple relationships between TO derivatives and cells
are being investigated.
Table 6
Reaction time and physical properties of compound 2a–2e
Entry
Yield (%)
Time (h)
Mp (°C)
2a
2b
2c
2d
2e
89
92
98
95
78
1
4
2
3
3
39
Liquid
67–69
74–76
105–108

590
X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
Table 7
(s, 1H), 7.31 (d, J = 6.90 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.73 (t,
Reaction time and yields of compound 3a–3e
J = 7.35 Hz, 1H), 7.77 (d, J = 9.00 Hz, 1H), 7.87 (s, 1H), 7.98 (d,
J = 9.00 Hz, 1H), 8.14 (d, J = 9.30 Hz, 1H), 8.77 (t, J = 7.95 Hz, 2H).
ESI-MS: m/e 377.33 (M+), 378.35 (M++1), 379.36(M++2).
Entry
Time (h)
Yield (%)
3a
3b
3c
3d
3e
24
24
24
48
96
81
92
33
53
21
4.12. TO derivative (5h)
Yield: 92%. mp: 206–208 °C. 1H NMR (300 MHz, DMSO-d6): d
2.44 (t, J = 6.60 Hz, 2H), 3.68 (t, J = 6.45 Hz, 2H), 4.04 (s, 3H), 4.75
(t, J = 6.90 Hz, 2H), 6.94 (s, 1H), 7.11 (d, J = 7.80 Hz, 1H), 7.50 (d,
J = 8.10 Hz, 1H), 7.80 (t, J = 7.05 Hz, 1H), 8.05 (t, J = 7.65 Hz, 1H),
8.18–8.22 (m, 2H), 8.50 (s, 1H), 8.78–8.86 (m, 2H). ESI-MS: m/e
456.42 (M+), 458.33 (M++2), 459.33 (M++3), 460.31 (M++4).
J = 8.10 Hz, 1H), 7.75 (d, J = 7.80 Hz, 1H), 7.80 (d, J = 8.70 Hz, 1H),
8.00 (t, J = 7.35 Hz, 1H), 8.07 (d, J = 7.80 Hz, 1H), 8.14 (d,
J = 8.70 Hz, 1H), 8.60 (d, J = 7.20 Hz, 1H), 8.81 (d, J = 8.40 Hz, 1H).
ESI-MS: m/e 411.66 (M+–1), 413.66 (M++1), 414.36 (M++2).
4.6. TO derivative (5b)
4.13. TO derivative (5i)
Yield: 95%. mp: 191–193 °C. 1H NMR (300 MHz, DMSO-d6): d
2.35 (t, J = 5.85 Hz, 2H), 2.98 (s, 3H), 3.12(t, J = 5.40 Hz, 2H), 5.18
(t, J = 6.75 Hz, 2H), 7.01 (t, J = 7.65 Hz, 1H), 7.10 (d, J = 9.00 Hz,
1H), 7.29 (t, J = 7.80 Hz, 1H), 7.52 (d, J = 9.00 Hz, 1H), 8.03 (t,
J = 7.65 Hz, 2H), 8.26 (t, J = 7.95 Hz, 1H), 854 (d, J = 9.00 Hz, 1H),
8.67 (d, J = 9.00 Hz, 1H), 9.40 (d, J = 6.00 Hz, 1H). ESI-MS: m/e
348.33 (M+), 349.26 (M++1), 350.25 (M++2).
Yield: 92%. mp: 182–184 °C. 1H NMR (300 MHz, DMSO-d6): d
2.91 (t, J = 7.05 Hz, 2H), 4.04 (s, 3H), 4.89 (t, J = 6.90 Hz, 2H), 6.97
(s, 1H), 7.12 (d, J = 8.40 Hz, 1H), 7.48 (d, J = 8.10 Hz, 2H), 8.22–
8.27 (m, 2H), 8.48 (s, 1H), 8.78–8.86 (m, 3H). ESI-MS: m/e 408.38
(M+), 409.40 (M++1), 410.39 (M++2).
4.14. TO derivative (5j)
Yield: 94%. mp: 228–230 °C. 1H NMR (300 MHz, DMSO-d6): d
2.45 (t, J = 7.65 Hz, 2H), 3.68 (t, J = 6.6 Hz, 2H), 4.01 (s, 3H), 4.80
(t, J = 6.75 Hz, 2H), 7.02 (s, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.50 (d,
J = 8.10 Hz, 2H), 7.83–7.87 (m, 2H), 8.08 (t, J = 7.35 Hz, 1H), 8.25
(d, J = 8.40 Hz, 1H), 8.86 (d, J = 7.20 Hz, 2H). ESI-MS: m/e 457.21
(M+), 458.21 (M++1), 459.22 (M++2), 460.21 (M++3).
4.7. TO derivative (5c)
Yield: 97%. mp: 171–173 °C. 1H NMR (300 MHz, DMSO-d6): d 2.74
(t, J = 6.45 Hz, 2H), 3.99 (s, 3H), 4.74 (t, J = 6.10 Hz, 2H,), 6.88 (s, 1H),
7.32 (d, J = 6.90 Hz, 1H), 7.40 (t, J = 7.65 Hz, 1H), 7.60 (t, J = 7.80 Hz,
1H), 7.75–7.78 (m, 2H), 7.96 (t, J = 7.8 Hz, 1H), 8.00 (d, J = 7.8 Hz,
1H), 8.12 (d, J = 9.00 Hz, 1H), 8.69 (d, J = 9.00 Hz, 1H), 8.77 (d,
J = 9.00 Hz, 1H). ESI-MS: m/e 363.25 (M+), 364.25 (M++1).
4.15. TO derivative (5k)
Yield: 93%. mp: 238–240 °C. 1H NMR (300 MHz, DMSO-d6): d
2.97 (t, J = 6.15 Hz, 2H), 4.02 (s, 3H), 4.91 (t, J = 6.15 Hz, 2H), 7.04
(s, 1H), 7.52 (d, J = 6.90 Hz, 1H), 7.85 (t, J = 9.00 Hz, 2H), 8.08 (t,
J = 7.40 Hz, 1H), 8.29 (d, J = 9.00 Hz, 1H), 8.42 (d, J = 8.70 Hz, 1H),
8.89 (t, J = 8.10 Hz, 2H), 9.00 (d, J = 8.10 Hz, 1H). ESI-MS: m/e
408.40 (M+), 409.42 (M++1), 410.39 (M++2).
4.8. TO derivative (5d)
Yield: 96%. mp: 150–152 °C. 1H NMR (300 MHz, DMSO-d6): d
2.39 (t, J = 6.70 Hz, 2H), 2.42 (s, 3H), 3.66 (t, J = 6.45 Hz, 2H), 4.00
(s, 3H), 4.66 (t, J = 7.20 Hz, 2H), 6.87 (s, 1H), 7.28 (d, J = 7.20 Hz,
1H), 7.42 (d, J = 8.70 Hz, 1H), 7.74 (d, J = 7.50 Hz, 1H), 7.68 (d,
J = 8.70 Hz, 1H), 7.84 (s, 1H), 7.98 (t, J = 7.65 Hz, 1H), 8.09(d,
J = 8.40 Hz, 1H), 8.54 (d, J = 9.00 Hz, 1H), 8.76 (d, J = 9.00 Hz, 1H).
ESI-MS: m/e 445.86 (M+ꢀ1), 447.48 (M++1), 449.42 (M++3).
4.16. Modification of TO–NH2 with folic acid
Folic acid (500 mg dissolved in 10 ml of dry dimethyl sulfoxide
(DMSO) plus 0.25 ml of Et3N was reacted with N-hydroxysuccini-
mine in the presence of dicyclohexylcarbodiimide overnight at
room temperature. The byproduct, dicyclohexylurea, was removed
by filtration. The filtrate was concentrated under reduced pressure
and the residue was precipitated in diethyl ether. The product, N-
hydroxysuccinimide eater of folic acid (NHS–folate) was washed
several times with anhydrous diethyl ether, dried under vacuum,
and stored as a yellow powder.17,31
Equal amount of NHS–folate and TO–NH2 were dissolved in
DMSO, adding Et3N to adjust pH to be weak basic. The mixture
was stirred at room temperature until the reaction was completed
by TLC. Precipitation with diethyl ether was followed by filtration
and yielded folate–TO–NH2.
4.9. TO derivative (5e)
Yield: 95%. mp: 207–210 °C. 1H NMR (300 MHz, DMSO-d6): d
2.47 (s, 3H), 2.65 (t, J = 6.90 Hz, 2H), 3.99 (s, 3H), 4.80 (t,
J = 5.10 Hz, 2H), 6.85 (s, 1H), 7.33 (d, J = 7.80 Hz, 2H), 7.37 (d,
J = 8.70 Hz, 1H), 7.57 (d, J = 8.10 Hz, 1H), 7.73 (d, J = 9.00 Hz, 2H),
7.96 (d, J = 7.80 Hz, 1H), 8.08 (d, J = 8.40 Hz, 1H), 8.70 (t,
J = 6.00 Hz, 2H). ESI-MS: m/e 397.41 (M+), 398.41 (M++1), 399.41
(M++2), 400.51 (M++3).
4.10. TO derivative (5f)
Yield: 96%. mp: 190–192 °C. 1H NMR (300 MHz, DMSO-d6): d
2.43 (t, J = 6.90 Hz, 2H), 3.66 (t, J = 6.30 Hz, 2H), 3.99 (s, 3H), 4.73
(t, J = 6.75 Hz, 2H), 6.92 (s, 1H), 7.37–7.47 (m, 2H), 7.78 (t,
J = 7.65 Hz, 1H), 7.93 (s, 1H), 8.04 (d, J = 9.00 Hz, 2H), 8.17 (d,
J = 9.00 Hz, 1H), 8.68 (d, J = 9.00 Hz, 1H), 8.82 (d, J = 9.00Hz, 1H).
ESI-MS: m/e 427.22 (M++1), 428.24 (M++2), 429.27 (M++3).
4.17. Modification of TO–COOH with CTS
H2O (8 mL) solution containing chitosan oligosaccharide
(600.00 mg, Mw < 3000) and DMSO (4.00 mL) solution containing
N,N-diisopropylethylamine (DIEA) (56 mg, 0.40 mmol) were dropped
into a 20 mL dimethylformamide (DMF) solution containing TO–
COOH (20 mg, 0.037 mmol), O-benzotriazole-N,N,N0,N0-tetramethyl-
uronium-hexafluorophosphate (HBTu) (41.20 mg, 0.109 mmol) and
N-hydroxybenzotriazole (HOBt) (14.72 mg, 0.109 mmol) which was
4.11. TO derivative (5g)
Yield: 95%. mp: 209–211 °C. 1H NMR (300 MHz, DMSO-d6): d
2.30 (t, J = 6.50 Hz, 1H), 3.93 (s, 1H), 4.74 (t, J = 5.85 Hz, 2H), 6.84

X. Fei et al. / Bioorg. Med. Chem. 17 (2009) 585–591
591
cooled at ꢀ5 °C, thereaction mixturewas stirredatroomtemperature
Acknowledgments
for 48 h, extracted with ether and ethyl acetate in turn after being
washed with acetone to give a floccule, which was isolated by filtra-
tion followed by washing with acetone and drying to afford a rose so-
lid TO–COOH–CTS (606.80 mg).
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 20672080), Key Project of Science and
Technology, Ministry of Education of China (Grant No. 205013),
Key Project of Science and Technology Committee of Tianjin (Grant
No. 08JCZDJC18200).
4.18. Modification of TO–COOH–CTS with folic acid
H2O (8 mL) solution containing TO–COOH–CTS (624.20 mg)
synthesized above and DMSO (1.00 mL) solution containing DIEA
(14 mg, 0.10 mmol) were dropped into a 5 mL DMF solution con-
taining folic acid (4.08 mg, 0.00925 mmol), HBTu (10.3 mg,
0.025 mmol) and HOBt (3.88 mg, 0.025 mmol) which was cooled
at ꢀ5 °C, the reaction mixture was stirred at room temperature
for 3 h. The mixture was extracted to give a floccule, and then
washed and dried to afford a rose solid folate–TO–COOH–CTS
(620.12 mg).
References and notes
1. Wang, L. Q.; Peng, X. J. Dyestuff Ind. (in Chinese) 2002, 39, 8.
2. Lin, Y.; Weissleder, R.; Tung, C. H. Bioconjugate Chem. 2002, 13, 605.
3. Timcheva, I. I.; Maximova, A. V.; Deligeorgiev, G. T.; Gadjev, I. N.; Sabnis, W. R.;
Ivanov, G. I. FEBS Lett. 1997, 405, 141.
4. Timtcheva, I. I.; Maximova, A. V.; Deligeorgegiev, G. T. J. Photochem. Photobiol.
2000, 58, 130.
5. Cai, Y.; Pailthorpe, M. T.; David, S. K. Textile Res. J. 1999, 69, 440.
6. Skeidsvoll, J.; Ueland, P. M. Anal. Biochem. 1995, 231, 359.
7. Timtcheva, I.; Maximova, V.; Deligeorgiev, T.; Zaneva, D.; Ivanov, I.
J. Photochem. Photobiol. A: Chem. 2000, 130, 7.
8. Yarmoluk, S. M.; Kryvorotenko, D. V.; Balanda, A. O., et al Dyes Pigments 2001,
51, 41.
9. Nygren, J.; Svanvi, N.; Kubista, M. Biopolymers 1998, 46, 39.
10. Fei, X. N.; Liu, L. J.; Zhang, B. L., et al Prog. Chem. 2006, 18, 801.
11. Rye, H. S.; Yue, S.; Wemmer, D. E., et al Nucleic Acids Res. 1992, 20, 2803.
12. Huang, J.; Wang, S. L.; Sun, X. Y., et al Polym. Bull. 2006, 81, 65.
13. Du, L. P.; Xiao, D. G. Chem. Ind. Times (in Chinese) 2005, 19, 31.
14. Benniston, A. C.; Harriman, A.; McAvoy, C. J. Chem. Soc., Faraday Trans. 1998, 94,
519.
15. Kim, E. M.; Jeong, H. J.; Park, I. K., et al J. Nucl. Med. 2005, 46, 141.
16. Weitman, S. D.; Weinberg, A. G.; Coney, L. R., et al Cancer Res. 1992, 52, 6078.
17. Moon, W. K.; Lin, Y. H.; O’Loughin, T.; Tang, Y.; Kim, D. E.; Weissleder, R.; Tung,
C. H. Bioconjuate Chem. 2003, 14, 539.
Folate–TO–COOH–CTS could also be synthesized by adding TO–
COOH, CTS and folic acid simultaneity to react by the same method
above.
4.19. Method of labeling cells
Different dye was dissolved in DMSO and saved as the stock
solutions respectively. The specific method of dyes labeling breast
cancer MCF-7 cell line was shown as below:
(1) MCF-7 cells were cultivated by routine method and the cul-
ture medium were minimum essential medium (MEM) sup-
plemented with 10% (v/v) of fetal bovine serum (FBS) and
18. Jhaveri, S. M.; Rait, S. A., et al Am. Assoc. Cancer Res. 2004, 3, 1505.
19. Paulos, M. C.; Reddy, A. J.; Christopher, P. Mol. Pharmacol. 2004, 166, 1406.
20. Morshed, K. M.; Ross, D. M.; McMartin, K. E. J. Nutr. 1997, 127, 1137.
21. Lee, R. J.; Low, P. S. Liposome Res. 1997, 7, 455.
´
´
Dulbeccos modified Eagles medium (DMEM) supplemented
22. Gabizon, A.; Catane, R.; Uziely, B., et al Cancer Res. 1994, 54, 987.
23. Sudimack, J.; Lee, R. J. Adv. Drug Delivery Rev. 2000, 41, 147.
24. Ross, J. F.; Chaudhuri, P. K.; Ratnam, M. Cancer 1994, 73, 2432.
25. Toffoli, G.; Cernigoi, C.; Russo, A.; Gallo, A.; Bagnoli, M.; Boiocci, M. Int. J. Cancer
1997, 74, 193.
26. Kennedy, M. D.; Jallad, K. N.; Thompson, D. H.; Ben-Amotz, D.; Low, P. S. J.
Biomed. Opt. 2003, 8, 636.
27. Tang, P. K. Chemistry and Technology of Fine Organic Chemistry; Tianjin
University Press: Tianjin, 1996. p 59.
28. Zhang, L.; Mizumoto, K.; Sato, N., et al Cancer Lett. 1999, 142, 129.
29. Teppema, J.; Sebrell, L. B. J. Am. Chem. Soc. 1927, 49, 1748.
30. Fei, X. N.; Yang, S. B.; Zhang, B. L.; Liu, Z. J.; Gu, Y. C. J. Comb. Chem. 2007, 9, 943.
31. Lee, J. R.; Low, S. P. J. Biol. Chem. 1994, 269, 3198.
with 10% (v/v) of FBS.
(2) The cells were transplanted to a 24-well plate and the cell
concentration was about 1 ꢁ 105/mL for each well. After
24 h, the old culture media was replaced with Hanks’ bal-
anced salt solution (HBSS). TO–COOH was added to con-
tinue to be incubated for 8 h at 37 °C under a 5% CO2
atmosphere.
(3) The cells were washed by HBSS three times and fluorescence
microscopy was performed using an inverted fluorescence
microscope.