Development of benzothiazole 'click-on' fluorogenic dyes

Article abstract of DOI:10.1016/j.bmcl.2010.11.009

'Click-on' fluorogenic reaction: a non-fluorescent benzothiazole with an electron-deficient alkyne group at 2-position reacts with azide containing molecules could form fluorescent adducts.

Full text of DOI:10.1016/j.bmcl.2010.11.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 320–323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of benzothiazole ‘click-on’ fluorogenic dyes
⇑
Jianjun Qi, Ching-Hsuan Tung
Department of Radiology, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX 77030, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
‘Click-on’ fluorogenic reaction: a non-fluorescent benzothiazole with an electron-deficient alkyne group
at 2-position reacts with azide containing molecules could form fluorescent adducts.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 September 2010
Revised 29 October 2010
Accepted 1 November 2010
Available online 5 November 2010
Keywords:
Fluorogenic dye
Benzothiazole
‘Click-on’ reaction
To study specific molecules in cells, precise labeling is the key.
Ideally, the fluorescent reporter should only tag the interested tar-
gets. Fluorescent proteins co-expressed with targeted molecules
have been widely used in studying cellular events, and the fluores-
cent signal makes the real time microscopic visualization of pro-
cess possible.1,2 However, the fluorescent proteins are large
proteins, which could be too big for many applications. Small pep-
tide tags have also been introduced into protein expressing sys-
tems for site-specific labeling.3,4 For instance, a peptide tag
recognized by Texas-red dye has been expressed in cells for fluo-
rescent calcium sensing.5 Tetracysteine (-Cys-Cys-Pro-Gly-Cys-
Cys-) containing proteins, which could be labeled by fluorogenic
biarsenical probes, have been developed for cell labels.6,7 Yet these
methods are still protein based approaches, which cannot be
applied to investigate non-protein molecules. In order to study
other biomolecules, flexible non-protein based labeling approaches
are required.
Theoretically, chemical approaches with millions of diversities
should be handy for intracellular labeling of biomolecules. Never-
theless, site-specific chemical labeling of biomolecules is not an
easy task. Reactions that require non-physiological conditions,
such as heat, extreme pH, or organic solvents, cannot be applied
to normal cells. Commonly used conjugating groups, including
activated ester which react with amino groups and maleimide
which react with sulfhydryl groups, these groups are not practical
for the site-specific labeling, due to high numbers of amino and
sulfhydryl groups in cells. A special chemoselective ligation reac-
tion has to be designed for each type of target. A few chemical liga-
tion approaches using ketone, azide, and alkyne as reactive groups
have been reported for cell labels as well.8 The typical procedure is
started by introducing a small percentage of chemically modified
building units together with normal building units, such as amino
acids, monosaccharides, or nucleosides, into cell culture. If the ap-
pended chemical group was small enough and could be tolerated
by natural synthase, the biosynthetic machinery would assemble
the biomacromolecules containing those unusual building units.
The introduced chemical reactive groups presented on biomacro-
molecules then would serve as the linking points for subsequent
fluorescent tagging.
In recent years, ‘click reaction’ has become a handy tool used in
drug discovery,9–11 combinatorial chemistry,12 material science,13
and tagging of biomolecules.14 The 1,2,3-triazole formation from
an azide and an alkyne can be done in aqueous condition, therefore
this approach has been applied in cells15,16 and, most recently, in
animals.17 Fluorescent reporters have been clicked onto almost
all kinds of biomolecules, such as nucleic acids, proteins, carbohy-
drates, and lipids. However, most fluorescent reporters used in
click chemistry are prepared from existing fluorochromes which
are functionalized with an alkyne or azide group through a linker.
Although the azide/alkyne click reaction is specific, excess fluoro-
chromes are always added to the reaction to ensure good conver-
sion. Since those reporters fluoresce at all time, high background
fluorescence signals prohibit real time visualization. Recently,
few fluorogenic dyes, such as coumarin 1 and 2,12,18 anthracene
319 and naphthalimide 4 derivatives,20 were reported to have no-
fluorescent property until the click reaction occurred. These
‘click-on’ fluorogenic dyes will not have the aforementioned high
background issue, because they are designed to be non-fluorescent
in their initial structure (Fig. 1). They only fluoresce after the com-
pletion of click reaction; therefore, the excess unreacted dyes will
not interfere with fluorescence signal.
⇑
Corresponding author. Tel.: +1 713 441 8682; fax: +1 713 441 8696.
E-mail address: ctung@tmhs.org (C.-H. Tung).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.009

J. Qi, C.-H. Tung / Bioorg. Med. Chem. Lett. 21 (2011) 320–323
321
property.21–24 Based on the chemical structure of benzothiazole
ring, it was hypothesized that an electron-deficient alkyne or
electron-rich azide groups at the 2-position would diminish the
fluorescence property. However, a click reaction which adds a con-
jugated triazole ring onto the parent benzothiazole ring will con-
vert the chromophore into a fluorophore.
Benzothiazole alkyne 9 was prepared using 6 as the starting
material (Scheme 1). Following the protocol developed by Elena
et al., 6 was transformed into iodobenzothiazole 7 by a diazotiza-
tion–iodination in the presence of p-toluenesulfonic acid in aceto-
nitrile.25 The protected acetylenic benzothiazole 8 was obtained by
the cross-coupling reaction of iodobenzothiazole 7 with a terminal
alkyne using catalytic quantities of Pd(PPh3)2Cl2 and CuI under ba-
sic conditions.26 The prepared 8 was then applied to a silica gel col-
umn for further purification; whereas it was found that the
protecting trimethylsilanyl group came off automatically during
chromatographic purification, resulting in the desired benzothia-
zole alkyne 9. As expected, 9 whose absorbance maximum is
286 nm (Fig. S1) is non-fluorescent.
O
O
O
O
R
N3
N
S
COOH
N
2
1
O
N
O
S
HO
5
3 N
3
4
Figure 1. Examples for existing fluorogenic dyes and luciferin 5.
Recognizing the usefulness of the switchable dyes, we have
developed a new benzothiazole based fluorogenic ‘click-on’ dye
and demonstrated its potential usages in nucleic acid and protein
labeling.
Benzothiazole alkyne 9 was chosen as the profluorophore since
its analog luciferin
5 has been shown to have fluorescent
N
S
N
S
N
N
c
a
b
SiMe3
NH2
I
S
S
9
6
8
7
Scheme 1. Reagents and condition: (a) p-MeC6H4SO3H, MeCN, rt to 10 °C, KI, NaNO2, H2O, 10 min, 10–15 °C; (b) (1) CuI, PdCl2(PPh3)2, NEt3, DCM, (2) NaHCO3, H2O; (c) acid.
CuSO4, sodium ascorbate, TBTA,
N
S
N
S
R
N
S
DMF/H2O(3/1), RT, 30min
N
N
N
N
or
N3
R
+
N
N
R
or CuI, DMF/MeOH,
100ºC, 12h
10
(a-d)
11
9
(a-d)
O
N
O
N
O
O
H3C
H3C
R: H
NH
NH
R:
Si
OH
OH
a
c
d
c
O
O
a
O
O
HO
HO
O
O
H
H
H
H
OH
OH
NHBoc
H
H
H
H
NHBoc
H
H
b
b
d
Scheme 2. Click reaction.
1.2
1.2
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
absorption
emission
absorption
emission
250
300
350
400
wavelength (nm)
Figure 2. Absorption and fluorescence of dye 16 (left) and 11a (right) in MeOH.
450
500
550
250
300
350
400
450
500
550
-0.2
-0.2
wavelength (nm)

322
J. Qi, C.-H. Tung / Bioorg. Med. Chem. Lett. 21 (2011) 320–323
Table 1
dine) (10b), 6-azido-hexanoic acid (10c) and Boc-p-azido-Phe-OH
(10d). Near quantitative conversion was obtained for all reactions
(Table S1), and all clicked products are fluorescent as compound
11a. In general, linking different groups to the 2-position of the
1,2,3-triazole ring has little effect on the absorption and emission
maxima (Table 1). All ‘click-on’ products (11a–d and 16) showed
absorption maximum at around 278–297 nm, emission maximum
at around 351–374 nm and stokes’ shift around 70–80 nm in meth-
anol. The fluorescence change of the ‘click-on’ reaction was moni-
tored in real time by reacting compound 9 (U = 0.14% in water,
UV absorption and emission spectra of compounds 11a–d and 16
11a
11b
11c
11d
16
kmax(abs) [nm]
kmax(em) [nm]
286
363
278
351
290
353
294
354
297
374
Condition: in MeOH, excitation wavelength: 285 nm.
To confirm that the proposed ‘click-on’ reaction can create a
fluorophore, the benzothiazole alkyne 9 was first reacted with
trimethylsilylazide 10a (according to method by Jin et al.)27 in
DMF/MeOH using CuI as catalyst, at 100 °C for 12 h (Scheme 2).
The reaction conversion was only around 20%. But importantly,
the clicked product 11a was fluorescent, having excitation and
emission at 285 nm and 363 nm, respectively, in methanol (Fig. 2
(right)). Later, refer to method by Chan et al.,28 TBTA (tris-(ben-
zyltriazolylmethyl)amine) was used as the catalyst under room
temperature for 1 h, the same reactions were tested with other
azide containing analogs, including nucleoside AZT (azidothymi-
using L-tryptophen as a reference) with 10d (U = 3.5%, in water)
catalyzed in an aqueous solution of sodium ascorbate and CuSO4
at rt. The broad emission band was observed from approximately
310 nm to 500 nm, which is characteristic of 11d (Fig. S2), once
the CuSO4 was added to the mixture solution of 9 and 10d in
1 mM NaAsc aqueous solution. As the reaction proceeds, the fluo-
rescence emission increases strongly. The fluorescent signal in-
creased from 1.6 a.u. to 254 a.u. within 24 h, representing a 158-
fold increase in fluorescence signal (Fig. 3).
O
O
OH
NHBoc
S
N
H2O, NaAsc, CuSO4, RT
OH
NHBoc
S
N
+
N
N
N3
N
9
10d
11d
nonfluorescent
fluorescent
300
24h
23h
22h
12h
11h
10h
9h
8h
7h
6h
5h
250
200
150
100
50
4h
3h
2h
1h
0h
0
310
360
410
460
wavelength/nm
Figure 3. Fluorescence emission spectra increase of a 3 ml H2O solution containing 9 (10
l
M), 10d (10
lM), NaAsc (1 mM) and CuSO4 (10
lM) (kex = 285 nm) within 24 h at
rt.
N
N
N
N
S
S
N
a
b
N
S
N
N N
N2X
NH2
S
N
13
12
c
14
6
NaN3
N
S
N
N
S
N
I
N
7
cis 13a
Scheme 3. Reagents and condition: (a) p-MeC6H4SO3H, MeCN, 0–10 °C, NaN3, NaNO2, H2O, 10 min, 10–15 °C; (b) NaN3; (c) NaN3, MeCN.

J. Qi, C.-H. Tung / Bioorg. Med. Chem. Lett. 21 (2011) 320–323
323
N
S
Supplementary data
N
S
º
CuI,DMF,Cs2CO3,120 C, 40h
N
HN
I
+
N
N
N
N
Supplementary data associated with (experimental details of
synthesis and characterization of the products) this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.11.009.
15
16
7
Scheme 4. Preparation of compound 16.
The above results showed that the electron deficient alkyne at
position 2 of the benzothiazole ring is critical to the ‘click-on’ reac-
tion. Nevertheless, the ‘click-on’ reaction is bi-directional, it is
interesting to see if an electron-rich azide group at the same posi-
tion could have a similar ‘click-on’ effect. Photoinduced electron
transfer (PET) effect by lone pair electrons has been widely docu-
mented in various fluorochromes,29 To synthesize benzothiazole
azide 13, two different approaches, direct diazotization of the com-
pound 6 and substitution of an iodol group on benzothiazole 7
with sodium azide, were tried. However none of them could lead
to the desired compound 13. The compound 13 seemed not sta-
ble,20 but forming the byproduct 14 through a spontaneous cycli-
zation as previously suggested (Scheme 3).30 Since the chemical
property didn’t allow isolating the ‘click-on’ compound 13, the ex-
pected final clicked product was prepared instead to validate the
hypothesis. The clicked product 16 was synthesized by reacting
iodobenzothiazole (7) with 1,2,3-trizole (15) referencing a re-
ported protocol (Scheme 4).24 Interestingly the compound 16 ex-
cites at 297 nm and emits at 374 nm (Fig. 2 (left)). Comparing
the structures of the compounds 16 and 11a, the substitution posi-
tions on the 1,2,3-trizole ring are different, but the conjugating
double bonds inside of 1,2,3-trizole ring of both compounds are ex-
tended over the benzothiazole ring, resulting in similar fluorescent
property. Although the ‘click-on’ compound 13 was not obtained in
this preparation, the compound 16 has suggested that the fluores-
cent property of an electron rich azide group containing dye could
also be activated by an alkyne group through a ‘click-on’ reaction.
Inconclusion, wedescribethefirst development of benzothiazole
‘click-on’ fluorogenic dyes. The click-on dye could have an electron-
rich azide group or an electron-deficient alkyne group at the 2-posi-
tion.Thekeyrequisitiontoachievethe‘click-on’fluorescentproperty
is that the newly formed double bonds inside of the 1,2,3-trizole ring
have to form conjugate double bonds with the parent fluorophore.
Potentially,thesameprinciplecouldbeextendedtodesignotherfluo-
rescence dyes. The demonstrated model reactions with functional-
ized nucleoside and amino acid have indicated that the developed
click-on dye could be applied to label various biomolecules, such as
nucleic acids, proteins and other molecules.
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We thank the NMR facility at M. D. Anderson Cancer Center for
acquiring all NMR data. This research was supported in part by NIH
CA135312, and CA114149.
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