Synthesis of hemicyanine dyes for 'click' bioconjugation

Article abstract of DOI:10.1016/j.tetlet.2005.01.066

A new type of 2-propynyl substituted hemicyanine dyes have been synthesized in a facile route, which have showed superior solubility in water and good optical properties. The terminal alkynyl groups were employed for bioconjugation through a 1,3-dipolar cycloaddition with azides under mild conditions.

Full text of DOI:10.1016/j.tetlet.2005.01.066

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1691–1695
Synthesis of hemicyanine dyes for ‘click’ bioconjugation
Wen-hai Zhan,a, Hannah N. Barnhill,b, Krishnamoorthy Sivakumar,b
He Tiana,* and Qian Wangb,*
aInstitute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, PRChina
bDepartments of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA
Received 14 November 2004; revised 12 January 2005; accepted 13 January 2005
Available online 29 January 2005
Abstract—A new type of 2-propynyl substituted hemicyanine dyes have been synthesized in a facile route, which have showed
superior solubility in water and good optical properties. The terminal alkynyl groups were employed for bioconjugation through
a 1,3-dipolar cycloaddition with azides under mild conditions.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
duce positive charges on the exterior surface of BNP
since they usually have good solubility in water, which
is crucial for the bioconjugation reaction. Cy3, Cy5,
and other cyanine based fluorescent analogs are among
the most commonly used bio-labeling fluorescent re-
agents.11–15 Since only traditional methods for the
attachment of cyanine dyes to biomolecules have been
exploited so far, such as the reaction of N-succinimidyl
ester for amino groups,16–18 or iodoacetamide for thiol
groups,19,20 the synthesis of bio-labeling cyanine dyes
in general was quite challenging. Herein, we describe
the synthesis of a novel type of water soluble alkyne
substituted hemicyanine dyes I–V (Scheme 1), which
could be successfully conjugated to biomolecules via
the ÔclickÕ bioconjugation reaction.
Huisgen 1,3-dipolar cycloaddition of azides and alkynes
to afford 1,2,3-triazole ring has been widely used in or-
ganic synthesis.1 The high reaction yield, simple reaction
and purification conditions, and the tolerance of other
functionalities of this reaction make it an ideal proto-
type to demonstrate the concept of ÔClick ChemistryÕ
developed by Sharpless and co-workers.2,3 The new
Fokin–Sharpless–Meldal modification, in which Cu(I)
is used as the catalyst, affords superior regioselectivity
and almost quantitative transformation under extremely
mild conditions.4,5 It accelerates the application of this
Ôclick processÕ in medicinal chemistry and biomedical re-
search.6 Alkyne and azide groups are very small in size,
are highly energetic with particularly narrow distribu-
tion of reactivity, can be conveniently introduced into
organic compounds, and are quite indifferent to solvent
and pH. Therefore, the Cu(I) catalyzed cycloaddition
reaction can be used to link biomolecules in an aqueous
environment under mild physiological conditions with
high efficiency and chemoselectivity.7–10
2. Results and discussion
Similar to our previous work in the synthesis of laser-
used hemicyanine dyes,21–23 the synthesis of compounds
I–V was very straightforward (Scheme 2). Since the ter-
minal alkyne group is inert to all reaction conditions
adopted in our synthesis, no additional protection and
deprotection steps were necessary. The synthesis of
I–III was started from 2-methylbenzothiazole X,
which was converted to XI by refluxing with propargyl
bromide. Condensation of XI with 4-N,N-dimethyl-
aminobenzaldehyde or 4-N,N-di-(2-carboxyethyl)amino-
benzal-dehyde or 4-N,N-dimethylaminocinnamaldehyde
resulted in dyes I–III. The dyes IV and V were synthe-
sized analogously starting from 1,1,2-trimethyl-1
H-benzoindole. All the dyes show superior solubility in
In our study of the interfacial self-assembly of bionano-
particles (BNPs), we hope to investigate how the surface
charges of BNP will influence the self-assembly process.
Cyanine and hemicyanine dyes are ideal regents to intro-
Keywords: Click reaction; Hemicyanine dye; 1,3-Dipolar cycloaddi-
tion; Bioconjugation; Cowpea mosaic virus.
*
Corresponding authors. Tel.: +1 803 777 8436; fax: +1 803 777
9521; e-mail addresses: wang@mail.chem.sc.edu; tianhe@ecust.edu.cn
These authors have equal contribution.
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.066

1692
W. Zhan et al. / Tetrahedron Letters 46 (2005) 1691–1695
S
S
S
CH2CH2COOH
N
N
N
N
N
N
CH2CH2COOH
Br
Br
Br
I
III
II
CH2CH2COOH
N
N
N
N
CH2CH2COOH
Br
Br
IV
V
HO
O
O
HO
O
O
N
N
COOH
COOH
H
N
HN
N
N3
O
O
HN
S
S
N
N3
N
H
O
O
O
O
O
O
H
VI
VII
VIII
IX
Scheme 1. Structures of fluorescent dyes used in ÔclickÕ bioconjugation.
R
OHC
Br
N
I. n = 0, R = CH3
S
n
S
R
II. n = 0, R = CH2CH2COOH
III. n = 1, R = CH3
N
N
CH3CN, reflux
20%
ethanol, reflux
63 - 98%
Br
X
XI
R
Br
OHC
N
IV. R = CH3
R
CH3CN, reflux
24%
ethanol, reflux
41 - 65%
N
V. R = CH2CH2COOH
N
Br
XII
Scheme 2. Synthetic routes of the hemicyanine dyes.
XIII
water, and have a strong absorbance as well as a large
Strokes shift of the fluorescence emission maxima (Table
1). The spectral properties of I and II, as well as IV and
V, are very similar to each other due to the structural
similarity.
Table 1. Absorption and fluorescent emission properties in 0.1 M
potassium phosphate buffer (pH 7.0) for dyes I–V
Dye
Absorption kmax/nm
e/Mꢀ1 cmꢀ1
Emission kmax/nm
I
533
530
553
578
571
90,460
61,370
69,330
55,000
54,700
588
593
680
604
605
II
III
IV
V
To test the reactivity in bioconjugation, icosahedral
cowpea mosaic virus (CPMV) was applied as a bio-
molecular prototype. CPMV has been demonstrated as

W. Zhan et al. / Tetrahedron Letters 46 (2005) 1691–1695
1693
a robust platform for many organic reactions.24
A
a catalytic system including CuSO4 as the copper source,
either a copper wire or tris(carboxyethyl)phosphine
(TCEP) as the reducing agent, and tris(triazolylamine)
as the ligand.7 Using a similar protocol (Scheme 3), we
first prepared the azide modified CPMV (Wt-CPMV-
N3),7 which was then conjugated with I–V catalyzed
by CuSO4 (1 mM), TCEP (2 mM) and tris(triazol-
yl)amine XIV (2 mM). After 15 h incubation at 4 ꢁC
and purification over gel filtration column, the products
were analyzed with UV–vis spectrometer and transmis-
sion electron microscopes. While dyes I, III, and IV de-
graded all the viral particles, dyes II and V afford high
recovery yield of intact viral particles (more than 60%)
and high conjugation efficiencies (>80%). Figure 1
number of functional groups and biomolecules have
been attached to the native reactive lysines and geneti-
cally inserted cysteines of CPMV.25–28 CPMV can be
made inexpensively on the gram scale and it is very easy
to separate the chemical modified viral particles from
unreacted small reagents. Therefore, it is an ideal model
system to test new bioconjugation reactions. In previous
reports, fluorescent compounds (VI–IX) were used to
study the influences of pH value, copper source, reduc-
ing reagent, copper concentration, and ligand structure
in copper(I) catalyzed 1,3-dipolar cycloaddition of
azides and alkynes.7,29 A standard ÔClickÕ protocol for
bioconjugation applications has been established using
EDC, Sulf-NHS
H
N
H
N
O
O
pH 7.0, 4oC, 24 h
(NH2)60
N3
+
HO
N
H
N3
n
O
O
Ultracentrifuge
65%
Wt-CPMV-N3
Wt-CPMV
Dye
(2) Size-exclusion
(1)
Chromatography
CuSO4 (1mM), XIV (2mM)
TCEP (2 mM), pH = 8.0, 4oC, 16 h
60%
Bn
N
N N
Dye
N
HO
HO
XIV
=
N
O
P
OH
TCEP
=
N
H
N
H
N
N
N
N
N
O
N
Bn
O
Bn
O
40
N
N
O
Scheme 3. Bioconjugation of alkyne substituted fluorescent dyes to CPMV by Cu(I) catalyzed 1,3-dipolar cycloaddition reaction.
Figure 1. UV–vis absorbance spectrum of CPMV modified with V. The peak of 260 nm is contributed by virus and the peak around 570 nm is
contributed by attached dye. (inset) The transmission electron microscope picture of CPMV-V conjugates after negative staining. The scale bar is
100 nm.

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W. Zhan et al. / Tetrahedron Letters 46 (2005) 1691–1695
shows the UV–vis absorbance spectrum and negative
staining transmission electron microscopy pictures of
V modified CPMV. The only structural difference be-
tween I and II (or IV and V) is the terminal carboxyl
groups. When CPMV (2 mg/mL) was incubated with
I–V (1.0 mM) alone, non-specific binding between dyes
and CPMV was not observed and intact viral particles
could be collected in high recover yield in all situations.
Therefore, the degradation of the viral particles when
click with I, III, and IV was caused by the reaction.
Since the reaction system was much more complicated
than a simple catalytic cycloaddition,7 many factors
could interfere the stability of the viral particles, which
will be of great interest for future investigation.
J = 8.9 Hz, 2H), 7.80 (t, J = 8.0 Hz, 1H), 7.7 (m, 2H);
6.90 (d, J = 9.0 Hz, 2H), 5.80 (s, 2H), 3.75 (m, 5H),
2.55 (m, 4H); HR-MS for C24H23N2O4S+ (the cation):
calculated 435.1379, found 435.1370. Dye III: mp
1
174 ꢁC; H NMR(DMSO- d6) d 8.33 (d, J = 7.8 Hz, 1
H), 8.16 (d, J = 7.6 Hz, 1H), 8.07 (m, 1H), 7.81 (t,
J = 7.6 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.58 (d,
J = 8.9 Hz, 2H), 7.51 (d, J = 14.9 Hz, 1H), 7.34 (d,
J = 14.4 Hz, 1H), 7.21 (m, 1H), 6.80 (d, J = 9.0 Hz, 2
H), 5.63 (d, J = 2.2 Hz, 2H), 3.75 (t, J = 2.4 Hz, 1H),
3.05 (s, 6H). Dye IV: mp 175–177 ꢁC; 1H NMR
(DMSO-d6) d 8.50 (d, J = 15.4 Hz, 1H), 8.35 (d,
J = 8.9 Hz, 1H), 8.22 (d, J = 8.9 Hz, 1H), 8.13 (m,
3H), 7.96 (d, J = 8.9 Hz, 1H), 7.75 (t, J = 7.3 Hz, 1H),
7.63 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 15.4 Hz, 1H), 6.92
(d, J = 9.0 Hz, 2H), 5.62 (d, J = 1.7 Hz, 2H), 3.65 (s,
1H), 3.20 (s, 6H), 1.98 (s, 6H); HR-MS for C27H27N2þ
(the cation): calculated 379.2175, found 379.2174. Dye
In conclusion, water soluble hemicyanine dyes I–V can
be prepared in a facile manner and could be used for
bioconjugation reactions under mild ÔclickÕ reaction
conditions. These reagents may also be used in labeling
other biologically important molecules. Compared to
common functionalities for bioconjugation, such as
N-succinimide ester, isothiocyanate, or maleimides,
the terminal alkyne group used in this study is more
synthetically friendly. Further modification and
improvement of the hemicyanine dyes structures in or-
der to afford better spectral and physical properties are
under exploitation.
1
V: H NMR(DMSO- d6) d 8.55 (d, J = 16.4 Hz, 1H),
8.34 (d, J = 9.8 Hz, 1H), 8.25 (d, J = 9.4 Hz, 1H), 8.14
(t, J = 8.2 Hz, 3H), 8.01 (d, J = 8.8 Hz, 1H), 7.76 (t,
J = 8.2 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.43 (d,
J = 15.7 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2 H), 5.62 (s,
2H), 3.80 (s, 2H), 3.67 (s, 2H), 3.33 (t, J = 2.38, 1H),
2.66 (t, J = 7.2 Hz, 1H), 2.59 (t, J = 6.5 Hz, 2H), 2.07
(s, 3H), 2.00 (s, 3H), 1.18 (t, J = 7.1 Hz, 2H). HR-MS
for C31H31N2O4 (the cation): calculated 495.2297, found
495.2284.
3. Typical experimental procedures
3.1. Synthesis of the dye I–V
3.2. General procedure of ‘click reactions’ on derivatized
viruses
7
Virus conjugates WT-CPMV-N3 (200 lg) and dye-al-
3.1.1.
2-[2-(4-Dimethylamino-phenyl)-vinyl]-3-prop-2-
kyne (0.25 mmol) were added to a solution of tris(triaz-
olyl)amine XIV (2 mM) and TCEP (2 mM) in a mixture
of 0.1 M potassium phosphate buffer (pH 8.0, 80 lL)
and DMF (20 lL) at 4 ꢁC. CuSO4 (0.5 M, 0.2 lL) was
then added. Following incubation at 4 ꢁC for 16 h, the
mixture was purified by passage through a P-100ꢂ (Bio-
Rad) size exclusion column (centrifugation at 800g for
6 min). This filtration was repeated with fresh columns
until all the excess reagents were removed (typically
three times). The recovery of derivatized viruses was
usually about 90%. All such samples were composed
of >95% intact particles (determined by sucrose gradient
ultra-sedimentation and TEM). Virus concentrations
were determined by measuring the absorbance at
260 nm; virus at 0.1 mg/mL gives a standard absorbance
of 0.8. The average molecular weight of the CPMV vir-
ion is 5.6 · 106 Da. The dye concentrations were ob-
tained by measurement of absorbance at the maximum
absorption wavelength with the new calibrated molar
absorptivity.
ynyl-benzothiazol-3-ium bromide (I). 2-Methylbenzo-
thiazole (12.0 g, 0.1 mol) was added into a solution of
propargyl bromide (5.0 g, 34 mmol) in acetonitrile
(20 mL) under vigorously stirring. Then the reaction
mixture was refluxed for 24 h. After cooling to room
temperature, the precipitation was collected to afford
the product XI as a gray solid (5.6 g, 20% yield); mp
1
224–226 ꢁC. H NMR(DMSO- d6) d 8.5 (d, J = 8.1 Hz,
1H), 8.38 (d, J = 8.5 Hz, 1H), 7.95 (t, J = 8 Hz, 1H),
7.83 (t, J = 7.8 Hz, 1H), 5.78 (d, J = 2.5 Hz, 2H), 3.85
(t, J = 2.5 Hz, 1H), 3.27 (s, 3H). The solution of 4-
N,N-dimethylaminobenzaldhyde (0.9 g, 6 mmol) and
XI (1.6 g, 6 mmol) in ethanol (45 mL) was refluxed for
2 h. Then the solvent was removed and the residue
was purified by column chromatography over silica gel
using dichloromethane/methanol (9:1, v/v) as eluents.
The product was collected as a dark purple solid
(2.0 g, 81% of yield); mp 180–181 ꢁC. 1H NMR
(DMSO-d6) d 8.30 (d, J = 7.8 Hz, 1H), 8.15 (m, 2H),
7.92 (d, J = 8.8 Hz, 2H), 7.80 (t, J = 8.0 Hz, 1H), 7.67
(m, 2H), 6.85 (d, J = 9.0 Hz, 2H), 5.75 (d, J = 2.2 Hz,
2H), 3.71 (t, J = 2.3 Hz, 1H), 3.1 (s, 6H); 13C NMR
(DMSO-d6) d 172.1, 154.6, 152.4, 141.1, 134.1, 129.7,
128.2, 127.4, 124.8, 122.2, 116.3, 112.8, 105.9, 78.9,
76.8, 38.3; HR-MS for C20H19N2S+ (the cation): calcu-
lated 319.1269, found 319.1269.
Acknowledgements
Q.W. would like to thank USA DOD Breast Cancer Re-
search Program, University of South Carolina Research
Foundation, USC Nanocenter for financial support.
This work was partially supported by Shanghai Munici-
pal Education Commission, PRChina.
3.1.2. Dye II. Mp 143–145 ꢁC; 1H NMR(DMSO- d6) d
8.30 (d, J = 8.1 Hz, 2H), 8.15 (m, 2H), 7.91 (d,

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