New indodicarbocyanine dyes for the biological microchip technology

Article abstract of DOI:10.1134/s1068162008010184

New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biologica

Full text of DOI:10.1134/s1068162008010184

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 1, pp. 130–133. © Pleiades Publishing, Inc., 2008.
Original Russian Text © V.E. Kuznetsova, V.A. Vasiliskov, O.V. Antonova, V.M. Mikhailovich, A.S. Zasedatelev, A.V. Chudinov, 2008, published in Bioorganicheskaya Khimiya,
2
008, Vol. 34, No. 1, pp. 141–144.
LETTERS
TO THE EDITOR
New Indodicarbocyanine Dyes
for the Biological Microchip Technology
V. E. Kuznetsova, V. A. Vasiliskov, O. V. Antonova, V. M. Mikhailovich,
1
A. S. Zasedatelev, and A. V. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received June 25, 2007; in final form, July 5, 2007
Abstract—New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment
bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most
effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence
maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount
of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The rela-
tive fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of
6
35 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.
Key words: biological microchips, fluorescence, hybridization analysis, indodicarbocyanine dyes
DOI: 10.1134/S1068162008010184
Fluorescent labeling has become one of the basic contains methyl groups in positions 5 and 7. This
techniques in various methods of the genetic analysis results in the bathochromic shift of absorption and flu-
[
1]. Fluorescent dyes should meet special requirements. orescence maxima. The reactive carboxybutyl group is
Fluorescent labels, providing high sensitivity and accu- in position 3 of the indolenine fragment (scheme). Such
racy of the instrumental data registration, should not structure provides a certain spatial orientation the fluo-
interfere with biochemical process of the test. When rofore relative to the oligonucleotide and decreases
using labels in near IR range, the self fluorescence of steric hindrances. The location and the total amount of
biological samples is low, thus increasing the sig- sulfonic groups make it possible to vary the total charge
nal/background ratio of the label and, therefore, raising of the dye and its water solubility.
the sensitivity of experimental data registration. Label-
Indodicarbocyanines of various structures are
ing with cyanine dyes is often used in the technology of
obtained by the condensation the corresponding indole-
biological microchips [2–4]. In this case, the test results
ninium bases (Ia)–(Ih) and malonic dialdehyde dianil
are registered on a biochip fluorescent analyzer sup-
hydrochloride (II) in two steps (scheme) [5–7]. Indole-
plied with industrial semiconductor laser light sources
ninium salt (IV) containing the carboxybutyl group was
with a wavelength of 635 (20 mW) and 655 nm
prepared from p-tolylhydrazine chloride and 6-methyl-
(
40 mW), providing registration of fluorescence at 670
7
-oxooctanoic acid by the Fisher cyclization and subse-
and 690 nm, respectively.
quent quaternization with 1,4-butanesulfone [7].
Cyanine dyes with the reactive carboxyl group in
positions 1 and 5 of the indolenine cycle had previously
been reported [5–7]. The goal of this study was the syn-
thesis of fluorescent dyes with absorption and the fluo-
rescence maxima being the most appropriate and pro-
viding the maximal sensitivity of registration.
The first step of indocarbocyanine was carried out
by heating in acetic anhydride or in acetic anhydride–
acetic acid. Diisopropylethylamine or anhydrous potas-
sium acetate was used as the condensing agent at the
second step [7].
The methods for the synthesis of different dyes vary
in the order of reagent mixing and the ratio of solvents.
All indodicarbocyanines synthesized were isolated by
We have suggested and carried out the synthesis of
a new series of indocarbocyanine dyes (Va)–(Vh)
(
scheme). Compounds (Va)–(Vh) are characterized by
reversed phase HPLC with a gradient of 0
50%
a number of distinctive features including water solu-
bility. The indolenine fragment of the dye molecule
acetonitrile in 0.05 M triethylammonium acetate buffer
solution. The yields of indocarbocyanine derivatives
varied from 5 to 50% depending on the compound
structure. The structure of the intermediates and the tar-
1
Corresponding author; phone: +7 (495) 135-9950; fax: +7 (495)
1
35-1405; e-mail: chud@eimb.ru.
1
30

NEW INDODICARBOCYANINE DYES
131
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 1 2008

1
32
KUZNETSOVA et al.
Spectral characteristics of indocarbocyanine dyes*
Relative efficiency of fluorescence
–
5
abc
max
em
max
ε × 10 ,
Compound
λ
, nm
λ
, nm
∆λ, nm
–1
–1
F
M cm
abc
em
λ
/ λ
655/690
635/670
max
max
(
Va)
649
51
650
51
653
54
654
55
651
52
651
52
656
54
654
55
665
16
17
16
19
17
20
17
20
15
18
17
20
20
18
17
21
2.1 ± 0.03
2.14 ± 0.03
2.17 ± 0.02
2.33 ± 0.02
1.96 ± 0.02
2.05 ± 0.02
1.95 ± 0.02
2.08 ± 0.01
1.73 ± 0.02
2.00 ± 0.01
1.71 ± 0.02
2.07 ± 0.02
1.82 ± 0.03
2.13 ± 0.03
1.49 ± 0.02
1.56 ± 0.02
0.10
0.14
0.12
0.18
0.09
0.16
0.10
0.15
0.08
0.12
0.09
0.14
0.07
0.11
0.06
0.09
0.492
0.736
0.664
1
0.104
0.276
0.181
0.373
0.185
0.442
0.216
0.456
0.098
0.239
0.120
0.342
0.164
0.382
0.071
0.151
0.313
0.469
0.400
0.604
0.263
0.409
0.261
0.367
0.222
0.349
0.222
0.392
0.172
0.292
0.097
0.180
6
668
666
670
670
674
671
675
666
670
668
672
676
672
671
676
(Vb)
6
(
Vc)
0.459
0.755
0.477
0.714
0.336
0.558
0.357
0.664
0.328
0.564
0.137
0.274
6
(Vd)
6
(
Ve)
6
(
Vf)
6
(Vg)
6
(Vh)
6
Note: * For each dye, the first line refers to the solution in PBS (10 mM potassium phosphate buffer solution, 0.9 % NaCl, pH 7.4); the
second, to the solution in MeOH.
1
get compounds was confirmed with UV and H NMR table). The highest values of these parameters have
spectroscopy and MALDI TOF mass spectrometry.
been found for dye (Vb).
The corresponding succinimide esters of (VIa)–
The relative fluorescence efficiency was measured
(
VIh) for labeling synthetic oligonucleotides (VII) at the equal concentrations of dyes (Va)–(Vh) in MeOH
bearing the amino linker with the C spacer in aqueous and in PBS in absorption and fluorescence maxima at
6
solutions were synthesized (scheme).
excitation wavelengths of 635 and 655 nm and registra-
tion wavelengths of 670 and 690 nm, respectively
The ultimate sensitivity of detection was determined
(
table).
Compound (Vd) is the most appropriate in sensitiv-
lyzer with a light source at 655 nm and registration at ity for aqueous solutions in a range of 655 nm/690 nm,
for oligonucleotides (VIIIa)–(VIIIh) labeled with dyes
(
Va)–(Vh). When using the biochip fluorescent ana-
6
1
90 nm, the ultimate sensitivity of detection was 3 × whereas compound (Vb) is the most appropriate in a
–18
0
mol.
range of 635 nm/670 nm.
Dyes (Va)–(Vh), when being used as labels for the
The dyes synthesized can be recommended for the
oligonucleotide primers, were found not to inhibit the use in biological microchip technology, in scientific
polymerase chain reaction (PCR). Labeled and the cor- research and medicinal diagnostics.
responding unlabeled primers participate in PCR with
equal efficiency.
ACKNOWLEDGMENTS
The molar extinction coefficient (ε) and the quan-
tum yield of fluorescence (F) in MeOH and aqueous
This work was supported in part by the U.S. Depart-
phosphate buffered saline (PBS, 10 mM potassium ment of Energy via the program Global Initiatives for
phosphate buffer, 0.9 % NaCl, pH 7.4) were found (the Proliferation Prevention (GIPP) at the support of the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 1 2008

NEW INDODICARBOCYANINE DYES
133
U.S. Civilian Research and Development Foundation
3. Harshman, K. and Martinez, A.C., European Review,
002, vol. 10, pp. 389–408.
2
(CRDF) and by the ISTC, grant no. 2906.
4
. Gerhold, D., Rushmore, T., and Caskey, C.T., Trends in
Biochemical Sciences, 1999, vol. 24, pp. 168–173.
5
. Mader, O., Reiner, K., Egelhaaf, H.-J., Fisher, R., and
Brock, R., Bioconjugate Chem., 2004, vol. 15, pp. 70–78.
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