Water-soluble cyanine dyes for biological microchip technology

Article abstract of DOI:10.1007/s11172-007-0387-3

Novel indodicarbocyanine dyes were obtained and their spectroscopic characteristics were determined. For equal concentrations of the dyes, the relative fluorescence efficiency was measured at the excitation wavelengths λ = 635 and 655 nm and the emission

Full text of DOI:10.1007/s11172-007-0387-3

Russian Chemical Bulletin, International Edition, Vol. 56, No. 12, pp. 2438—2442, December, 2007
2438
Waterꢀsoluble cyanine dyes for biological microchip technology
a
V. E. Kuznetsova,a T. A. Luk´yanova,a V. A. Vasiliskov,a O. V. Kharitonova,b A. V. Chudinov,a and A. S. Zasedatelev

aV. A. Engelgardt Institute of Molecular Biology, Russian Academy of Sciences,
32 ul. Vavilova, 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 1405. Eꢀmail: chud@eimb.ru
bM. V. Lomonosov Moscow State Academy of Fine Chemical Technology,
86 prosp. Vernadskogo, 119571 Moscow, Russian Federation.
Fax: +7 (495) 434 8711
Novel indodicarbocyanine dyes were obtained and their spectroscopic characteristics were
determined. For equal concentrations of the dyes, the relative fluorescence efficiency was
measured at the excitation wavelengths λ = 635 and 655 nm and the emission wavelengths
λ = 670 and 690 nm, respectively.
Key words: hybridization analysis, cyanine dyes, oligonucleotides, fluorescence.
Scheme 1
Hybridization analysis with fluorescence labeling is
widely used in biological microchip technology. A variety
of methodological techniques of analysis and recording of
results necessitates a search for novel efficient fluorescent
markers.
We proposed and conducted the synthesis of a novel
series of waterꢀsoluble fluorescent dyes 1a—h of the nearꢀ
IR range for labeled primers technique using a polymerase
chain reaction.1 Dyes 1a—h are characterized by the presꢀ
ence of a reactive carboxy group that is attached to posiꢀ
tion 3 of the indolenine fragment through the tetramethꢀ
ylene chain and is designed to label biomolecules. Beꢀ
cause of such a structure, the fluorophore part of the dye
is distant from a molecule to be labeled, which enhances
its ability to form specific complexes.
Indoleninium salt 2 containing the carboxybutyl group
was prepared from phenylhydrazine (3) and 6ꢀmethylꢀ7ꢀ
oxooctanoic acid2—6 (4) by the Fischer cyclization folꢀ
lowed by quaternization of indolenine 5 with 4ꢀhydroxꢀ
ybutaneꢀ1ꢀsulfonic acid7—9 (Scheme 1).
The synthesis of cyanine dyes 1a—h involves sequenꢀ
tial condensation reactions of appropriate indoleninium
salts (6a—h) with malonaldehyde dianil (7) and of interꢀ
mediate 8 with indoleninium salt 2 (Scheme 2).
Depending on indoleninium salt 6a—h added in the
first step, the synthesis of indodicarbocyanines was carꢀ
ried out either in acetic anhydride under heating or in
acetic anhydride—acetic acid.10—12 In the second step,
ethyl(diisopropyl)amine or anhydrous potassium acetate
was employed as a condensation agent.12 The use of anꢀ
hydrous sodium acetate sharply lowered the yield of the
target product.
All the indodicarbocyanines 1a—h obtained were isoꢀ
lated by reverse phase chromatography (RPꢀ18 column,
MeCN—0.05 M triethylammonium acetate buffer, gradiꢀ
ent elution from 0 to 50% MeCN) and converted into
sodium salts. The yields of the indodicarbocyanines varꢀ
ied from 5 to 48%. It should be noted that lithium salts of
dyes 1a—h are very unstable in storage. The structures of
intermediates and target products were confirmed by eleꢀ
mental analysis, electronic absorption spectroscopy, MALꢀ
DIꢀTOF mass spectrometry, and 1H NMR spectroscopy.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2355—2359, December, 2007.
1066ꢀ5285/07/5612ꢀ2438 © 2007 Springer Science+Business Media, Inc.

Waterꢀsoluble cyanine dyes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 12, December, 2007 2439
Scheme 2
1, 6, 8, 9, 11: R1 = Et, R2 = R3 = H (a); R1 = (CH2)4SO3–, R2 = R3 = H (b); R1 = Et, R2 = Me, R3 = H (c); R1 = (CH2)4SO3–, R2 = Me,
3 = H (d); R1 = Et, R2 = SO3–, R3 = H (e); R1 = (CH2)4SO3–, R2 = SO3–, R3 = H (f); R1 = Et, R2 = R3 = Me (g); R1 = (CH2)4SO3
,
–
R
R
2 = R3 = Me (h)
The indodicarbocyanines obtained have different absorpꢀ
lengths of 635 (20 mV) and 655 nm (40 mV) that provide
tion and fluorescence wavelengths, which was attained by
introducing additional methyl substituents into indoleniniꢀ
um salts 6a—h. The water solubility and total charge of the
dye depends on the number of sulfo groups in its molecule.
For all indodicarbocyanine dyes 1a—h, we determined
molar absorption coefficients and fluorescence quantum
yields in methanol and an aqueous phosphate salt buffer
(PBS) (0.01 M K3PO4, 0.9% NaCl, pH 7.4) (Table 1).
The sensitivity of fluorescence detection is determined
by the absolute sensitivity of a label defined as the product
of the molar absorption coefficient and the fluorescence
quantum yield. Thus, we determined the absolute detecꢀ
tion sensitivity for indodicarbocyanines 1a—h in terms of
the corresponding absorption and fluorescence peaks.
In biological microchip technology, analysis is carried
out on a fluorescence biochip analyzer fitted with comꢀ
mercial semiconducting laser light sources with waveꢀ
fluorescence at λ = 670 and 690 nm, respectively. We
measured the relative fluorescence efficiencies of dyes
1a—h at the above wavelengths (see Table 1). The results
obtained were normalized to the maximum value found
for dye 1b in methanol.
Succinimide esters 9a—h were obtained in quantitaꢀ
tive yields under the action of Nꢀhydroxysuccinimide in
the presence of dicyclohexylcarbodiimide in anhydrous
DMF.13 Synthetic oligonucleotides 10 containing an amiꢀ
no linker with a C6 spacer were labeled with activated
esters 9a—h in aqueous solutions (see Scheme 2).
Fluorescent oligonucleotides proved to be completely
suitable for labeled primers technique using a polymerase
chain reaction followed by hybridization on oligonucleꢀ
otide biochips. For aqueous solutions, the sensitivity of
dye 1a is best at λ = 655/690 nm and that of dye 1b is best
at λ = 635/670 nm.

2440
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 12, December, 2007
Kuznetsova et al.
Table 1. Fluorescence properties of the cyanine dyes*
abs
em
max
Comꢀ
pound
Solvent
λ
λ
ε•10–5
/L mol–1 cm–1
Φ
Relative fluorescence
max
efficiency**
nm
em
λ
abs/λ
655/690 635/670
max
max
1a
1b
1c
1d
1e
1f
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
643
644
647
648
647
649
649
651
645
646
646
648
650
652
650
651
657
662
661
664
662
667
665
669
660
664
660
665
665
670
668
669
2.06 0.02
2.47 0.02
2.43 0.02
2.40 0.02
1.99 0.03
2.01 0.01
2.16 0.02
2.29 0.01
2.17 0.02
2.29 0.03
2.20 0.02
2.43 0.02
1.84 0.01
1.93 0.01
1.46 0.02
1.54 0.02
0.13
0.25
0.17
0.27
0.09
0.17
0.11
0.19
0.14
0.27
0.14
0.25
0.08
0.12
0.05
0.08
0.444
0.881
0.677
1
0.183
0.030
0.102
0.222
0.081
0.178
0.121
0.260
0.087
0.225
0.096
0.283
0.082
0.165
0.039
0.092
0.147
0.714
0.427
0.681
0.220
0.382
0.283
0.471
0.325
0.673
0.325
0.706
0.158
0.251
0.057
0.109
0.312
0.528
0.428
0.667
0.491
0.830
0.522
0.958
0.238
0.375
0.082
0.167
1g
1h
MeOH
* ε is the molar absorption coefficient; Φ is the fluorescence quantum yield; PBS is a 0.01 M potassium phosphate
buffer, 0.9% NaCl, pH 7.4.
** The relative fluorescence efficiencies are given at different excitation/emission wavelengths (λ/nm).
1H NMR (CDCl3), δ: 0.56—0.81 (m,
2
H,
Experimental
CH2CH2CH2CH2COOH); 1.28 (s, 3 H, H3C(3)); 1.43—1.51
(m, 2 H, CH2CH2CH2CH2COOH); 1.76—1.92 (m, 2 H,
1
H NMR spectra were recorded on a Bruker AMXꢀ400 pulse
CH2CH2CH2CH2COOH);
2.13—2.21
(m,
2
H,
Fourier radiospectrometer (Germany) (400 MHz) in CDCl3,
D2O, and DMSOꢀd6 with Me4Si as the external standard. Mass
spectra were recorded on a Compact MALDI 4 MALDIꢀTOF
instrument (Kratos Analytical, USA). UV spectra were recorded
on a Jasco Vꢀ550 spectrophotometer (Japan). Fluorescence specꢀ
tra were recorded on a Shimadzu RF 5000 spectrofluorimeter
(Japan). Melting points were determined on a Kofler Boetius
hot stage (Germany). All solvents were purified to meet specific
requirements.
Column chromatography was carried out on Lichroprep
RPꢀ18 (particle size 0.040—0.063 nm; Merck, Germany) in a
column 10×200 mm.
Indolenine bases 6a—h,7,10,12,14, malonaldehyde dianil,15
and 6ꢀmethylꢀ7ꢀoxooctanoic acid (4)2—6 were prepared as deꢀ
scribed earlier.
3ꢀ(4ꢀCarboxybutyl)ꢀ2,3ꢀdimethylindolenine (5). A mixture of
6ꢀmethylꢀ7ꢀoxooctanoic acid (4) (720 mg, 4.2 mmol), phenylꢀ
hydrazine (3) (420 µL, 4.2 mmol), and glacial AcOH (4.2 mL)
was heated in an inert atmosphere at 118 °C for 3.5 h. The
solvent was removed and the oily residue was dissolved in chloꢀ
roform (10 mL). The resulting solution was washed with water
and brine and dried with MgSO4. The solvent was removed and
the residue was recrystallized from EtOAc—hexane (0.6 : 2).
The yield of indolenine 5 was 0.8 g (78%), a yellow orange
powder, m.p. 112—113 °C. Found (%): C, 73.49; H, 7.76;
N, 5.72. C15H19NO2. Calculated (%): C, 73.44; H, 7.81; N, 5.71.
UV (MeOH), λmax/nm: 256. MS, m/z 246.3 [M]+. C15H19NO2.
Calculated: M = 245.32.
CH2CH2CH2CH2COOH); 2.24 (s, 3 H, H3C(2)); 7.18—7.54
(m, 4 H, Ar).
3ꢀ(4ꢀCarboxybutyl)ꢀ2,3ꢀdimethylꢀ1ꢀ(4ꢀsulfonatoꢀ
butyl)indoleninium (2). A mixture of indolenine 5 (640 mg,
2.6 mmol), 4ꢀhydroxybutaneꢀ1ꢀsulfonic acid (530 µL,
5.2 mmol), and 1,2ꢀdichlorobenzene (4.2 mL) was heated at
118 °C for 25 h. The solvent was decanted and the oily residue
was triturated with diethyl ether. The precipitate that formed
was filtered off, washed with diethyl ether, and dried in a vacuꢀ
um desiccator over P2O5. The yield of indoleninium salt 2 was
0.98 g (99%), a dark cherry powder, m.p. 48—50 °C. Found (%):
C, 59.78; H, 7.16; N, 3.69. C19H27NO5S. Calculated (%):
C, 59.82; H, 7.13; N, 3.67. UV (MeOH), λmax/nm: 280. MS,
m/z 382.0 [M]+. C19H27NO5S. Calculated: M = 381.49.
1H NMR (D2O), δ: 0.38—0.68 (m,
CH2CH2CH2CH2COOH); 1.23—1.35 (m,
2
2
H,
H,
CH2CH2CH2CH2COOH); 1.43 (s, 3 H, H3C(3)); 1.71—1.81
(m, H, CH2CH2CH2CH2SO3); 1.92—2.20 (m, H,
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 2.84 (t, 2 H,
CH2CH2CH2CH2SO3, 7.5 Hz); 4.41 (t, H,
2
6
J
=
2
CH2CH2CH2CH2SO3, J = 7.5 Hz); 7.18—7.70 (m, 4 H, Ar).
3ꢀ(4ꢀCarboxybutyl)ꢀ1´ꢀethylꢀ3,3´,3´ꢀtrimethylꢀ1ꢀ(4ꢀsulfoꢀ
natobutyl)indodicarbocyanine (1a). A mixture of 1ꢀethylꢀ2,3,3ꢀ
trimethylindoleninium iodide (6a) (63 mg, 0.2 mmol), malonalꢀ
dehyde dianil hydrochloride (7) (52 mg, 0.2 mmol), acetic anꢀ
hydride (700 µL), and acetic acid (300 µL) was heated at 118 °C
for 2 h. Then the solvent was removed in vacuo. The residue (8)
was dissolved in acetic anhydride (1.7 mL). A solution of

Waterꢀsoluble cyanine dyes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 12, December, 2007 2441
3ꢀ(4ꢀcarboxybutyl)ꢀ2,3ꢀdimethylꢀ1ꢀ(4ꢀsulfonatobutyl)indoꢀ
leninium (2) (80 mg, 0.21 mmol) in a mixture of ethyl(diisoꢀ
propyl)amine (200 µL, 2.1 mmol) and acetic anhydride (800 µL,
7.2 mmol) was added. The reaction mixture was kept at ~20 °C
for a day. The reaction products were isolated by reverse phase
chromatography on an RPꢀ18 column in MeCN—0.05 M triꢀ
ethylammonium acetate buffer with gradient elution from 0 to
50% MeCN. The solvents were removed and the residue was
diluted with water, put again on the top of the RPꢀ18 column,
washed with 0.1 M NaCl and water, and isolated by reverse
phase chromatography in MeCN—H2O. The solvent was reꢀ
moved in vacuo and the residue was dried in a vacuum desiccaꢀ
tor over P2O5. The yield of dye 1a was 46 mg (37.5%), m.p.
172—173 °C. Found (%): C, 69.55; H, 7.28; N, 4.64.
C35H44N2O5S. Calculated (%): C, 69.51; H, 7.33; N, 4.63.
The fluorescence characteristics of the dye are given in Table 1.
MS, m/z 606.6 [M]+. C35H44N2O5S. Calculated: M = 604.80.
1H NMR (DMSOꢀd6), δ: 0.43, 0.78 (both m, 1 H each,
CH2CH2CH2CH2COOH); 1.24—1.31 (m, 5 H, CH2CH3,
CH2CH2CH2CH2COOH); 1.64 (s, 9 H, H3C(3), H3C(3´));
1H NMR (DMSOꢀd6), δ: 0.48, 0.78 (both m, 1 H each,
CH2CH2CH2CH2COOH); 1.22—1.35 (m, 5 H, CH2CH3,
CH2CH2CH2CH2COOH); 1.64 (s, 9 H, H3C(3), H3C(3´)); 1.73
(m,
4
H, CH2CH2CH2CH2SO3); 2.01 (m,
2.18 (m,
2
H,
H,
CH2CH2CH2CH2COOH);
1
CH2CH2CH2CH2COOH); 2.37 (s, 3 H, H3C(5´)); 2.45 (m,
3 H, CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 4.12
(m, 4 H, CH2CH3, CH2CH2CH2CH2SO3); 6.32 (m, 1 H, αꢀCH,
α´ꢀCH); 6.55 (t, 1 H, γꢀCH, J = 12.0 Hz); 7.21—7.55 (m, 7 H,
Ar); 8.27 (m, 2 H, βꢀCH, β´ꢀCH).
3ꢀ(4ꢀCarboxybutyl)ꢀ3,3´,3´,5´ꢀtetramethylꢀ1,1´ꢀbis(4ꢀsulfoꢀ
natobutyl)indodicarbocyanine, sodium salt (1d) was obtained as
described for compound 1a. The yield was 54 mg (36%), m.p.
120—122 °C. Found (%): C, 60.98; H, 6.55; N, 3.77.
C38H49N2NaO8S2. Calculated (%): C, 60.94; H, 6.59; N, 3.74.
For the fluorescence characteristics, see Table 1. MS,
m/z 726.9 [M]+. C38H49N2O8S2–. Calculated: M = 725.94.
1H NMR (DMSOꢀd6), δ: 0.47, 0.79 (both m, 1 H each,
CH2CH2CH2CH2COOH);
1.32
(m,
2
H,
CH2CH2CH2CH2COOH); 1.65—1.81 (s, 17 H, H3C(3),
1.73 (m,
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 4.12 (m,
4 H, CH2CH3, CH2CH2CH2CH2SO3); 6.26 (d, 1 H, α´ꢀCH,
J = 13.5 Hz); 6.41 (d, 1 H, αꢀCH, J = 13.5 Hz); 6.57 (t, 1 H,
γꢀCH, J = 12.0 Hz); 7.21—7.61 (m, 8 H, Ar); 8.32 (m, 2 H,
βꢀCH, β´ꢀCH).
4
H, CH2CH2CH2CH2SO3); 1.89 (m,
2
H,
H,
H,
H3C(3´), CH2CH2CH2CH2SO3); 2.03 (m,
2
H,
H,
2.18
2.41
(m,
(m,
1
3
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH); 2.36 (s,
CH2CH2CH2CH2COOH); 2.48
2.14
(m,
4
(m,
1
H, H3C(5´),
H,
4
CH2CH2CH2CH2SO3); 4.08 (m, 4 H, CH2CH2CH2CH2SO3);
6.35 (m, 2 H, αꢀCH, α´ꢀCH); 6.57 (t, 1 H, γꢀCH, J = 12.5 Hz);
7.19—7.54 (m, 7 H, Ar); 8.30 (m, 2 H, βꢀCH, β´ꢀCH).
3ꢀ(4ꢀCarboxybutyl)ꢀ1´ꢀethylꢀ3,3´,3´ꢀtrimethylꢀ5´ꢀsulfoꢀ
1ꢀ(4ꢀsulfonatobutyl)indodicarbocyanine, sodium salt (1e) was
obtained as described for compound 1b. The yield was 32 mg
(22%), m.p. 139—141 °C. Found (%): C, 59.51; H, 6.11;
N, 3.98. C35H43N2NaO8S2. Calculated (%): C, 59.47; H, 6.13;
N, 3.96. For the fluorescence characteristics, see Table 1. MS,
m/z 684.3 [M]+. C35H43N2O8S2–. Calculated: M = 683.86.
1H NMR (DMSOꢀd6), δ: 0.47, 0.77 (both m, 1 H each,
CH2CH2CH2CH2COOH); 1.24—1.38 (m, 5 H, CH2CH3,
CH2CH2CH2CH2COOH); 1.68 (m, 13 H, H3C(3), H3C(3´),
3ꢀ(4ꢀCarboxybutyl)ꢀ3,3´,3´ꢀtrimethylꢀ1,1´ꢀbis(4ꢀsulfonatoꢀ
butyl)indodicarbocyanine, sodium salt (1b). A mixture of 2,3,3ꢀ
trimethylꢀ1ꢀ(4ꢀsulfonatobutyl)indoleninium (6b) (59 mg,
0.2 mmol), malonaldehyde dianil hydrochloride (7) (52 mg,
0.2 mmol), acetic anhydride (700 µL), and acetic acid (160 µL)
was heated at 118 °C for 2 h. Then 3ꢀ(4ꢀcarboxybutyl)ꢀ2,3ꢀ
dimethylꢀ1ꢀ(4ꢀsulfonatobutyl)indoleninium (2) (80 mg,
0.21 mmol), anhydrous potassium acetate (117 mg, 1.2 mmol),
acetic anhydride (700 µL), and acetic acid (350 µL) were added
and the reaction mixture was heated at 118 °C for 2 h. Comꢀ
pound 1b was isolated as described for compound 1a. The yield
of dye 1b was 71 mg (48%), m.p. 184—186 °C. Found (%):
C, 60.41; H, 6.42; N, 3.84. C37H47N2NaO8S2. Calculated (%):
C, 60.47; H, 6.45; N, 3.81. For the fluorescence characteristics,
see Table 1. MS, m/z 712.1 [M]+. C37H47N2O8S2–. Calculated:
M = 711.91.
CH2CH2CH2CH2SO3);
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH);
2.00
2.19
2.40
(m,
(m,
(m,
2
1
3
H,
H,
H,
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 4.13 (m,
4 H, CH2CH3, CH2CH2CH2CH2SO3); 6.26 (d, 1 H, α´ꢀCH,
J = 13.5 Hz); 6.43 (d, 1 H, αꢀCH, J = 13.5 Hz); 6.57 (t, 1 H,
γꢀCH, J = 12.0 Hz); 7.23—7.79 (m, 7 H, Ar); 8.33 (m,
2 H, βꢀCH, β´ꢀCH).
1H NMR (DMSOꢀd6), δ: 0.45, 0.77 (both m, 1 H each,
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH); 1.65—1.80 (s, 17 H, H3C(3),
H3C(3´), CH2CH2CH2CH2SO3); 1.91 (m,
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH);
1.32
(m,
2
H,
3ꢀ(4ꢀCarboxybutyl)ꢀ3,3´,3´ꢀtrimethylꢀ5´ꢀsulfoꢀ1,1´ꢀbis(4ꢀ
sulfonatobutyl)indodicarbocyanine, disodium salt (1f) was obtained
as described for compound 1b. The yield was 54 mg (32%),
m.p. >250 °C. Found (%): C, 53.15; H, 5.51; N, 3.37.
C37H46N2Na2O11S3. Calculated (%): C, 53.10; H, 5.54;
2
H,
H,
H,
2.17
2.42
(m,
(m,
1
5
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 4.09 (m,
4 H, CH2CH2CH2CH2SO3); 6.37 (m, 2 H, αꢀCH, α´ꢀCH);
6.57 (t, 1 H, γꢀCH, J = 12.0 Hz); 7.21—7.6 (m, 8 H, Ar); 8.30
(m, 2 H, βꢀCH, β´ꢀCH).
N, 3.35. For the fluorescence characteristics, see Table 1.
MS, m/z 789.6 [M]+. C37H46N2O11S3
. Calculated:
2–
M = 790.97.
1H NMR (DMSOꢀd6), δ: 0.46, 0.78 (both m, 1 H each,
3ꢀ(4ꢀCarboxybutyl)ꢀ1´ꢀethylꢀ3,3´,3´,5´ꢀtetramethylꢀ1ꢀ(4ꢀ
sulfonatobutyl)indodicarbocyanine (1c) was obtained as described
for compound 1a. The yield was 40 mg (32%), m.p.
151—153 °C. Found (%): C, 69.91; H, 7.47; N, 4.55.
C36H46N2O5S. Calculated (%): C, 69.87; H, 7.49; N, 4.53. For
the fluorescence characteristics, see Table 1. MS, m/z 619.3
[M]+. C36H46N2O5S. Calculated: M = 618.83.
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH); 1.65—1.75 (s, 17 H, H3C(3),
H3C(3´), CH2CH2CH2CH2SO3); 2.02 (m,
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 4.09 (m,
4 H, CH2CH2CH2CH2SO3); 6.38 (m, 2 H, αꢀCH, α´ꢀCH);
1.33
(m,
2
H,
2
H,
H,
H,
2.16
2.39
(m,
(m,
1
5

2442
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 12, December, 2007
Kuznetsova et al.
6.59 (t, 1 H, γꢀCH, J = 12.0 Hz); 7.22—7.79 (m, 7 H, Ar); 8.32
(m, 2 H, βꢀCH, β´ꢀCH).
monium acetate buffer (0.5 mL) was added and the product was
extracted with butanꢀ1ꢀol until the upper layer decolorized comꢀ
pletely. Colored oligonucleotide 11a—h was isolated by HPLC.
Monitoring was carried out at λ = 254 nm, flow rate 1 mL
min–1, gradient elution from A—B (20%) to B (100%) over
30 min (A is a 0.1 M triethylammonium acetate buffer and B is a
50% solution of acetonitrile in A). The yield of labeled oligonuꢀ
cleotide was 75—80%. The structures of the conjugates obtained
were confirmed by MALDIꢀTOF mass spectrometry.
3ꢀ(4ꢀCarboxybutyl)ꢀ1´ꢀethylꢀ3,3´,3´,5´,7´ꢀpentamethylꢀ1ꢀ
(4ꢀsulfonatobutyl)indodicarbocyanine (1g) was obtained as deꢀ
scribed for compound 1a. The yield was 19 mg (20%), m.p.
110—112 °C. For the fluorescence characteristics, see Table 1.
MS, m/z 633.6 [M]+. C37H48N2O5S. Calculated: M = 632.85.
1H NMR (DMSOꢀd6), δ: 0.49, 0.78 (both m, 1 H each,
CH2CH2CH2CH2COOH); 1.33 (m,
5
H, CH2CH3,
CH2CH2CH2CH2COOH); 1.64 (s, 9 H, H3C(3), H3C(3´)); 1.72
This work was financially supported by the Internaꢀ
tional Scientific and Technical Center (ISTC Grant 2906).
(m,
4
H, CH2CH2CH2CH2SO3); 2.01 (m,
2.14 (m,
2
H,
H,
CH2CH2CH2CH2COOH);
1
CH2CH2CH2CH2COOH); 2.31 (s, 3 H, H3C(5´)); 2.4 (m, 3 H,
CH2CH2CH2CH2COOH, CH2CH2CH2CH2SO3); 2.59 (s, 3 H,
References
H3C(7´)); 4.06 (m,
2 H, CH2CH3); 4.27 (m, 2 H,
CH2CH2CH2CH2SO3); 6.35 (m, 1 H, αꢀCH, α´ꢀCH); 6.56 (d,
1 H, αꢀCH, J = 12.0 Hz); 6.99—7.54 (m, 6 H, Ar); 8.29 (m, 2 H,
βꢀCH, β´ꢀCH).
3ꢀ(4ꢀCarboxybutyl)ꢀ3,3´,3´,5´,7´ꢀpentamethylꢀ1,1´ꢀbis(4ꢀ
sulfonatobutyl)indodicarbocyanine, sodium salt (1h) was obtained
as described for compound 1b. The yield was 8 mg (5%), m.p.
108—110 °C. For the fluorescence characteristics, see Table 1.
MS, m/z 741.2 [M]+. C39H51N2O8S2–. Calculated: M = 739.96.
1H NMR (DMSOꢀd6), δ: 0.48, 0.79 (both m, 1 H each,
1. R. T. Ranasinghe and T. Brown, Chem. Commun., 2005,
5487.
2. P. J. Hamrick, Jr., C. F. Hauser, and C. R. Hauser, J. Org.
Chem., 1959, 24, 583.
3. ChungꢀLing Mao, F. C. Frostick, E. H. Man, R. M. Manyꢀ
ik, R. L. Wells, and C. R. Hauser, J. Org. Chem., 1969,
34, 1425.
4. R. M. Manyik, F. C. Frostick, Jr., J. J. Sanderson, and C. R.
Hauser, J. Am. Chem. Soc., 1953, 75, 5030.
CH2CH2CH2CH2COOH);
1.32
(m,
2
H,
5. H. K. Sen and U. Böse, Q. J. Indian Chem. Soc., 1927, 4, 51;
Chem. Zentralbl., 1927, 2, 435.
6. G. B. Payne, J. Org. Chem., 1961, 4793.
7. S. R. Mujumdar, R. B. Mujumdar, C. M. Grant, and A. S.
Waggoner, Bioconjugate Chem., 1996, 7, 356.
CH2CH2CH2CH2COOH); 1.67—1.81 (s, 17 H, H3C(3),
H3C(3´), CH2CH2CH2CH2SO3); 2.05 (m,
2
H,
H,
CH2CH2CH2CH2COOH);
CH2CH2CH2CH2COOH); 2.36 (s,
CH2CH2CH2CH2COOH); 2.47
2.18
(m,
4
1
H, H3C(5´),
H,
(m,
4
8. L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Wagꢀ
goner, Cytometry, 1989, 10, 3.
9. N. Narayanan and G. Patonay, J. Org. Chem., 1995, 60, 2391.
10. O. Mader, K. Reiner, H.ꢀJ. Egelhaaf, R. Fisher, and R.
Brock, Bioconjugate Chem., 2004, 15, 70.
11. A. J. G. Mank, H. T. C. van der Laan, C. Gooijer, U. A.
Brinkman, and N. H. Velthorst, Anal. Chem., 1995, 67, 1742.
12. R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis,
and A. S. Waggoner, Bioconjugate Chem., 1993, 4, 105.
13. P. L. Southwick, L. A. Ernst, E. W. Tauriello, S. R. Parker,
R. B. Mujumdar, S. R. Mujumdar, H. A. Clever, and A. S.
Waggoner, Cytometry, 1990, 11, 418.
14. P. L. Southwick, J. G. Cairns, L. A. Ernst, and A. S. Wagꢀ
goner, Org. Prep. Proc. Int., 1988, 20, 279.
15. L. A. Yanovskaya, S. S. Yufit, and V. F. Kucherov, Izv.
Akad. Nauk SSSR, Ser. Khim., 1960, 1246 [Bull. Acad. Sci.
USSR, Div. Chem. Sci., 1960, 9, 7 (Engl. Transl.)].
CH2CH2CH2CH2SO3); 2.61 (s, 3 H, H3C(7´)); 4.12 (m, 4 H,
CH2CH2CH2CH2SO3); 6.37 (m, 2 H, αꢀCH, α´ꢀCH); 6.59 (t,
1 H, γꢀCH, J = 12.5 Hz); 7.21—7.55 (m, 6 H, Ar); 8.33 (m, 2 H,
βꢀCH, β´ꢀCH).
Succinimide esters of indodicarbocyanine dyes 9a—h. NꢀHyꢀ
droxysuccinimide (69 mg, 0.6 mmol) and a solution of dicycloꢀ
hexylcarbodiimide (62 mg, 0.3 mmol) in anhydrous DMF
(500 µL) were added to a solution of a sodium salt of indodicarꢀ
bocyanine 1a—h (0.15 mmol) in anhydrous DMF (11 mL) cooled
to –18 °C. The mixture was stirred at ~20 °C for 5 days, diluted
with water, and filtered. The filtrate was put on the top of an
RPꢀ18 column, washed with water (100 mL), and eluted with
MeCN—water (1 : 1). The solvent was removed and the residue
was dried in a vacuum desiccator over P2O5. The yields of sucꢀ
cinimide esters 9a—h were 85—90%.
Oligonucleotide labeling. A 0.1 M carbonateꢀbicarbonate
buffer (15 µL) and acetonitrile (15 µL) were added to the oligoꢀ
nucleotide 5´ꢀCTCAGTTTꢀNH2ꢀ3´ (10) (200 nmol). The mixꢀ
ture was thoroughly stirred and cooled to –18 °C. The succinꢀ
imide ester of dye 9a—h (~0.6 mg) was added and the mixture
was stirred and kept at ~20 °C for 24 h. Then a 0.1 M triethylamꢀ
Received May 25, 2007;
in revised form August 2, 2007