Novel asymmetric indodicarbocyanine dyes

Article abstract of DOI:10.1007/s11172-007-0356-x

Novel indodicarbocyanine dyes functionalized at position 5 of the indolenine system were obtained and characterized by spectroscopic methods.

Full text of DOI:10.1007/s11172-007-0356-x

Russian Chemical Bulletin, International Edition, Vol. 56, No. 11, pp. 2263—2267, November, 2007
2263
Novel asymmetric indodicarbocyanine dyes
V. E. Kuznetsova, A. V. Davydov, V. A. Vasiliskov, A. A. Stomakhin, A. V. Chudinov,ꢀ and A. S. Zasedatelev
V. A. Engel´gardt Institute of Molecular Biology, Russian Academy of Sciences,
32 ul. Vavilova, 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 1405. Eꢀmail: chud@eimb.ru
Novel indodicarbocyanine dyes functionalized at position 5 of the indolenine system were
obtained and characterized by spectroscopic methods.
Key words: indodicarbocyanine dyes, indolenines, 4ꢀ(2ꢀcarboxyethyl)phenylhydrazine, fluoꢀ
rescence.
Highꢀintensity fluorescence of indodicarbocyanine
dyes in the nearꢀIR range (600—800 nm) makes them
most suitable markers for analysis of oligonucleotides and
proteins in biological microchip technology.1
their binding to the fluorophore also have a strong effect
on the properties of dyes and their complexes with
biomolecules and, consequently, on the whole bioꢀ
analysis.1,6
Indocyanine dyes with the carboxy group attached to
the indolenine fragment in position 1 (through a polyꢀ
methylene chain2—4) and position 5 (directly and through
a methylene unit5—8) have been documented. However,
these dyes have some drawbacks. A probe attached to the
carboxy and carboxymethyl groups in position 5 of the
indolenine ring is in close vicinity of the bulky fluorophore,
which prevents complete formation of specific complexes
between a compound to be analyzed and the fluorescent
probe. The nature of the substituents and the locations of
The goal of the present work was to obtain a novel
series of indodicarbocyanine dyes containing a 5ꢀcarboxyꢀ
ethylindolenine fragment. The synthesis of 5ꢀ(2ꢀcarboxyꢀ
ethyl)ꢀ2,3,3ꢀtrimethylindolenine (1) and its derivatives
2a,b is shown in Scheme 1.
4ꢀNitrocinnamonitrile (3) was synthesized9 from
4ꢀnitroaniline (4) through 2ꢀchloroꢀ3ꢀ(4ꢀnitropheꢀ
nyl)propionitrile (5). Hydrolysis of compound 3 in
conc. H3PO4 gave 4ꢀnitrocinnamic acid (6). Catalytic
hydrogenation of acid 6 on 5% Pd/C in AcOH followed
Scheme 1
R = Et•I– (a), (CH2)4SO3– (b)
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2186—2190, November, 2007.
1066ꢀ5285/07/5611ꢀ2263 © 2007 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 56, No. 11, November, 2007
Kuznetsova et al.
Scheme 2
16: R1 = Et, R2 = H (a); R1 = Et, R2 = SO3– (b); R1 = (CH2)4SO3–, R2 = H (c); R1 = (CH2)4SO3–, R2 = Me (d); R1 = (CH2)4SO3–, R2 = SO3– (e)
by hydrochlorination afforded 3ꢀ(4ꢀaminophenyl)propꢀ
ionic acid hydrochloride (7) in 96% yield. Diazotization
of compound 7 and reduction10 of the resulting diazoꢀ
nium salt with SnCl2•2H2O in HCl gave 4ꢀ(2ꢀcarboxyꢀ
ethyl)phenylhydrazine hydrochloride (8).
Indolenine 1 was obtained by the Fischer cyclization11
of compound 8 with isopropyl methyl ketone in AcOH in
the presence of AcOK without isolating intermediate hydrꢀ
azone. Alkylation of indolenine 1 with ethyl iodide or
4ꢀhydroxybutaneꢀ1ꢀsulfonic acid yielded the correspondꢀ
ing derivatives 2a,b.
Dyes 9—15 were obtained by twoꢀstep condensaꢀ
tion of appropriate indoleninium salts 16a—e and 2a,b
with malonaldehyde dianil hydrochloride (17) in Ac2O
(Scheme 2, Table 1).
The first step involved condensation of indolenines
16a—e (containing no carboxy group) with dianil hydroꢀ
chloride 17. Addition of acetyl chloride to the reaction
mixture largely lowered the fraction of the symmetric dye.
In the second step, indolenine 2a or 2b was added
and condensation was carried out in the presence of
ethyl(diisopropyl)amine or potassium acetate as conꢀ
Table 1. Spectroscopic characteristics of indodicarbocyanine dyes 9—15
abs
em
Comꢀ
pound
R1
R2
R3
Solvent
λ
λ
ε•10–5
/L mol–1 cm–1
Φ
max
max
nm
–
9
Et
Et
H
SO3
H
(CH2)4SO3
Et
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
PBS
MeOH
646
648
648
650
646
648
652
654
647
649
648
650
648
650
658
666
662
670
660
665
662
673
662
669
656
668
664
670
1.46 0.02
1.92 0.02
1.54 0.02
1.43 0.03
1.52 0.03
1.56 0.02
1.34 0.03
1.89 0.05
1.32 0.01
1.97 0.03
1.72 0.01
1.62 0.02
1.37 0.03
1.69 0.04
0.11
0.14
0.09
0.11
0.11
0.16
0.11
0.19
0.11
0.17
0.08
0.14
0.10
0.14
–
10
11
12
13
14
15
–
–
–
(CH2)4SO3
(CH2)4SO3
(CH2)4SO3
Et
Et
–
–
–
–
Me
H
(CH2)4SO3
(CH2)4SO3
(CH2)4SO3
(CH2)4SO3
–
SO3
SO3
–
–
(CH2)4SO3
Note. λ abs and λ em are the absorption and fluorescence peak wavelengths, respectively; ε is the molar absorption
max
max
coefficient; Φ is the fluorescence quantum yield; PBS is a 0.01 M solution of a potassium phosphate buffer,
0.9% NaCl, pH 7.4.

Indodicarbocyanine dyes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 11, November, 2007 2265
carbon under heating. The yield of 3ꢀ(4ꢀaminophenyl)propionic
acid hydrochloride (7) was 3.9 g (96%), m.p. 186—187 °C.
Found (%): C, 53.68; H, 5.95; N, 7.02. C9H12ClNO2. Calcuꢀ
lated (%): C, 53.61; H, 6.00; N, 6.95. 1H NMR (DMSOꢀd6), δ:
2.53 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 2.83 (t, 2 H,
CH2CH2COOH, J = 7.5 Hz); 7.28—7.35 (d, 4 H, Ar, J =
8.5 Hz); 10.31 (s, 2 H, NH2).
densation agents. All the dyes obtained were isolated
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 structures of all the compounds were
confirmed by elemental analysis data, MALDIꢀTOF mass
spectra, and 1H NMR spectra.
4ꢀ(2ꢀCarboxyethyl)phenylhydrazine hydrochloride (8).
A threeꢀneck flask fitted with a mechanical stirrer and a therꢀ
mometer was charged with 3ꢀ(4ꢀaminophenyl)propionic acid
hydrochloride (7) (2.0 g, 10 mmol) and conc. HCl (35 mL), and
the mixture was cooled. Then a solution of NaNO2 (0.7 g,
10 mmol) in water (0.9 mL) was added dropwise under the layer
of the liquid. The addition rate was such as to prevent a rise of
the reaction temperature above –5 °C. The mixture was warmed
to 0 °C for 30 min and then recooled to –5 °C. A solution of
SnCl2•2H2O (10 g, 44 mmol) in conc. HCl (15 mL) was added
precooled to –18 °C. The reaction temperature was maintained
at no higher than –5 °C by regulating the addition rate. The
mixture was additionally stirred at –5 °C for 30 min and kept at
5 °C for ~24 h. The crystals that formed were filtered off and
washed with conc. HCl (6 mL) cooled to –18 °C. The yield of
compound 8 was 1.62 g (75%), m.p. 191—192 °C. Found (%):
C, 49.95; H, 6.09; N, 12.87. C9H13ClN2O2. Calculated (%):
For dyes 9—15, we determined absorption and fluoꢀ
rescence peaks, molar absorption coefficients, and relaꢀ
tive quantum yields of fluorescence in MeOH and a phosꢀ
phateꢀsalt buffer (see Table 1). Dyes 9—15 have good
fluorescent characteristics, are thermally and chemically
stable, and are resistant to light under bioanalysis condiꢀ
tions. Because of this, they can be successfully used as
fluorescent markers in hybridization analysis. At present,
dyes 9—15 are being additionally tested through biologiꢀ
cal microchip technology.
Experimental
1
H NMR spectra were recorded on a pulse Bruker AMXꢀ400
1
Fourier spectrophotometer (Germany) (400 MHz) in CDCl3
(with Me4Si as the external standard) and DMSOꢀd6.
MALDIꢀTOF mass spectra were recorded on a Compact
MALDI 4 instrument (Kratos Analytical, USA).
C, 49.89; H, 6.05; N, 12.93. H NMR (DMSOꢀd6), δ: 2.47 (t,
2 H, CH2CH2COOH, J = 7.5 Hz); 2.73 (t, 2 H, CH2CH2COOH,
J = 7.5 Hz); 6.91—7.13 (d, 4 H, Ar, J = 8.5 Hz); 10.27 (s,
3 H, NHNH2).
UV spectra were recorded on a Jasco Vꢀ550 spectrophotoꢀ
meter (Japan); fluorescence spectra were recorded on a
Shimadzu RF 5000 spectrofluorimeter (Japan). Melting points
were determined on a Koffler 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 two
columns 16×400 mm and 12×100 mm.
5ꢀ(2ꢀCarboxyethyl)ꢀ2,3,3ꢀtrimethylindolenine (1). A mixture
of compound 8 (216 mg, 1 mmol), anhydrous AcOK (196 mg,
2.0 mmol), and 3ꢀmethylbutanꢀ2ꢀone (150 µL, 1.37 mmol) was
heated in acetic acid (1 mL) at 118 °C for 1 h. Then the solvent
was removed and the residue was dissolved in CHCl3, washed
with water and brine, and dried with Na2SO4. The solvent was
removed and the oily residue was recrystallized from ethyl acꢀ
etate—hexane (1 : 10). The yield of 5ꢀ(2ꢀcarboxyethyl)ꢀ2,3,3ꢀ
trimethylindolenine (1) was 118 mg (55%), m.p. 175—176 °C.
Found (%): C, 72.64; H, 7.39; N, 6.01. C14H17NO2. Calcuꢀ
lated (%): C, 72.70; H, 7.41; N, 6.06. UV (MeOH), λmax/nm:
262. MS, m/z: 232.3 [M]+. C14H17NO2. Calculated: M = 231.29.
1H NMR (CDCl3), δ: 1.27 (s, 6 H, 2 C(3)H3); 2.27 (s, 3 H,
C(2)H3); 2.68 (t, 2 H, CH2CH2COOH, J = 8 Hz); 3.00 (t, 2 H,
CH2CH2COOH, J = 8 Hz); 7.12—7.46 (d, 3 H, Ar, J = 8.5 Hz).
5ꢀ(2ꢀCarboxyethyl)ꢀ1ꢀethylꢀ2,3,3ꢀtrimethylindoleninium
iodide (2a). A mixture of compound 1 (230 mg, 1 mmol) and
ethyl iodide (500 µL, 5.5 mmol) was heated in acetonitrile (2 mL)
at 80 °C for 15 h. On cooling to ~20 °C, anhydrous diethyl ether
(5 mL) was added and the mixture was kept at –18 °C for 24 h.
The crystals that formed were filtered off and dried in a vacuum
desiccator over P2O5 and KOH. The yield of compound 2a was
377 mg (97%), m.p. 194—195 °C. Found (%): C, 49.68; H, 5.71;
N, 3.58. C16H22INO2. Calculated (%): C, 49.62; H, 5.73;
N, 3.62. UV (MeOH), λmax/nm: 284. MS, m/z: 259.8 [M]+.
C16H21NO2+. Calculated: M = 259.34. 1H NMR (DMSOꢀd6),
δ: 1.44 (t, 3 H, CH2CH3, J = 7.0 Hz); 1.52 (s, 6 H, 2 C(3)H3);
2.62 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 2.80 (s, 3 H, C(2)H3);
2.96 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 4.47 (q, 2 H,
CH2CH3, J = 7.0 Hz); 7.47—7.86 (m, 3 H, Ar).
Indolenine bases 16a—e,2,4,8 malonaldehyde dianil,12
2ꢀchloroꢀ3ꢀ(4ꢀnitrophenyl)propionitrile (5),8 and 4ꢀnitroꢀ
cinnamonitrile (3)8 were prepared as described earlier.
4ꢀNitrocinnamic acid (6). A mixture of 4ꢀnitrocinnamonitrile
(3) (17.4 g, 0.1 mol) and conc. H3PO4 (350 mL) was heated at
150 °C for 2 h. On cooling to ~20 °C, the mixture was poured
into ice water (4 L). The precipitate of crude product 6 (18.3 g)
was filtered off, dissolved in 0.12 M NaOH (1 L), and treated
with activated charcoal (3.5 g). The resulting solution was filꢀ
tered and the filtrate was acidified to pH ~2 by adding dropwise
conc. HCl. The precipitate that formed was filtered off, washed
with water, and dried at 90 °C. The yield of 4ꢀnitrocinnamic
acid (6) was 17.7 g (92%), yellow needles, m.p. >250 °C
(cf. Ref. 9: m.p. 283—284 °C). Found (%): C, 55.92; H, 3.71;
N, 7.28. C9H7NO4. Calculated (%): C, 55.96; H, 3.65; N, 7.25.
1H NMR (DMSOꢀd6), δ: 6.74 (d, 1 H, CHCHCOOH, J =
16.5 Hz); 7.69 (d, 1 H, CHCHCOOH, J = 16.5 Hz); 7.96—8.25
(d, 4 H, Ar, J = 9 Hz).
3ꢀ(4ꢀAminophenyl)propionic acid hydrochloride (7). A vigorꢀ
ously stirred mixture of the catalyst 5% Pd/C (400 mg), acetic
acid (80 mL), and 4ꢀnitrocinnamic acid (6) (3.9 g, 20 mmol)
was hydrogenated in a hydrogen flow at ~1 atm and ~20 °C for
2.5 h. The reaction mixture was filtered and the filtrate was
concentrated. The oily residue was dissolved in a mixture of
5 M HCl (7 mL) and water (30 mL) and treated with activated
5ꢀ(2ꢀCarboxyethyl)ꢀ2,3,3ꢀtrimethylꢀ1ꢀ(4ꢀsulfonatobutyl)inꢀ
doleninium (2b). A mixture of compound 1 (230 mg, 1 mmol)
and 4ꢀhydroxybutaneꢀ1ꢀsulfonic acid (160 µL, 1.6 mmol) was

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Kuznetsova et al.
heated in butyronitrile (2.2 mL) at 118 °C for 22 h. The reaction
mixture was cooled to ~20 °C and triturated with anhydrous
diethyl ether. The precipitate that formed was filtered off and
dried in a vacuum desiccator over P2O5 and KOH. The yield of
compound 2b was 300 mg (86%), m.p. >250 °C. Found (%):
C, 58.75; H, 6.92; N, 3.82. C18H25NO5S. Calculated (%):
C, 58.83; H, 6.86; N, 3.81. UV (MeOH), λmax/nm: 284. MS,
m/z: 368.2 [M]+. C18H25NO5S. Calculated: M = 367.46.
1H NMR (DMSOꢀd6), δ: 1.51 (s, 6 H, 2 C(3)H3); 1.73 (m, 2 H,
CH2CH2CH2CH2SO3); 1.96 (m, 2 H, CH2CH2CH2CH2SO3);
2.61 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 2.81 (s, 3 H, C(2)H3);
2.95 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 4.46 (m, 2 H,
CH2CH2CH2CH2SO3); 7.47—7.93 (m, 3 H, Ar).
J = 7.5 Hz); 4.95 (m, 4 H, CH2CH3); 6.30 (d, 2 H, αꢀCH,
α´ꢀCH, J = 13.5 Hz); 6.58 (t, 1 H, γꢀCH, J = 12.0 Hz);
7.21—7.63 (m, 6 H, Ar); 8.31 (m, 2 H, βꢀCH, β´ꢀCH).
5ꢀ(2ꢀCarboxyethyl)ꢀ1ꢀethylꢀ3,3,3´,3´ꢀtetramethylꢀ1´ꢀ
(4ꢀsulfonatobutyl)indodicarbocyanine (11). Compound 11 was obꢀ
tained as described for compound 10. The yield was 59 mg
(50%), m.p. 185—187 °C. Found (%): C, 69.15; H, 7.21; N, 4.71.
C34H42N2O5S. Calculated (%): C, 69.12; H, 7.17; N, 4.74. The
spectroscopic characteristics of the dye are given in Table 1.
MS, m/z: 592.8 [M]+. C34H42N2O5S. Calculated: M = 590.77.
1H NMR (DMSOꢀd6), δ: 1.28 (t, 3 H, CH2CH3, J = 7.0 Hz);
1.67 (s, 12 H, 2 C(3)H3, 2 C(3´)H3); 1.74 (m, 4 H,
CH2CH2CH2CH2SO3); 2.27 (t,
2 H, CH2CH2COOH,
5ꢀ(2ꢀCarboxyethyl)ꢀ1´ꢀethylꢀ3,3,3´,3´ꢀtetramethylꢀ1ꢀ
(4ꢀsulfonatobutyl)indodicarbocyanine (9). A mixture of compound
16a (54 mg, 0.2 mmol) and malonaldehyde dianil hydrochloride
(17) (59 mg, 0.23 mmol) was heated in acetic anhydride (1 mL)
containing acetyl chloride (64 µL) at 118 °C for 2 h. Then the
solvent was removed in vacuo and the residue was dissolved in
acetic anhydride (2 mL). Compound 2b (81 mg, 0.22 mmol)
and a mixture (550 µL) of ethyl(diisopropyl)amine (200 µL,
2.1 mmol) and acetic anhydride (800 µL, 7.2 mmol) were added.
The reaction mixture was kept at ~20 °C for 24 h. Reaction
products were prepurified by flash chromatography on an RPꢀ18
column. Then the target compound was isolated by reverse phase
chromatography on an RPꢀ18 column in MeCN—0.05 M
Et3NH+AcO– buffer by gradient elution from 0 to 50% MeCN.
The solvents were removed and the residue was diluted with
water, placed again in the RPꢀ18 column, and washed with
0.1 M NaCl and water. The product was 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 9 was 57 mg (48%), m.p. >250 °C.
Found (%): C, 69.18; H, 7.12; N, 4.78. C34H42N2O5S. Calcuꢀ
lated (%): C, 69.12; H, 7.17; N, 4.74. The spectroscopic charꢀ
acteristics of the dye are given in Table 1. MS, m/z: 591.0 [M]+.
J = 7.5 Hz); 2.48 (m, 2 H, CH2CH2CH2CH2SO3); 2.82
(t, 2 H, CH2CH2COOH, J = 7.5 Hz); 4.13 (m, 4 H,
CH2CH2CH2CH2SO3, CH2CH3); 6.30 (d, 2 H, αꢀCH, α´ꢀCH,
J = 13.5 Hz); 6.56 (t, 1 H, γꢀCH, J = 12.0 Hz); 7.24—7.58 (m,
7 H, Ar); 8.28 (m, 2 H, βꢀCH, β´ꢀCH).
5ꢀ(2ꢀCarboxyethyl)ꢀ3,3,3´,3´,5´ꢀpentamethylꢀ1,1´ꢀbis(4ꢀ
sulfonatobutyl)indodicarbocyanine, sodium salt (12). Compound
12 was obtained as described for compound 9. The yield was
81 mg (55%), m.p. 150—152 °C. Found (%): C, 60.51; H, 6.49;
N, 4.85. C37H47N2NaO8S2. Calculated (%): C, 60.47; H, 6.45;
N, 3.81. The spectroscopic characteristics of the dye are given in
Table 1. MS, m/z: 713.0 [M]+. C37H47N2O8S2–. Calculated:
M = 711.91. 1H NMR (DMSOꢀd6), δ: 1.66 (s, 12 H, 2 C(3)H3,
2 C(3´)H3); 1.74 (m, 8 H, CH2CH2CH2CH2SO3); 2.36 (s, 3 H,
C(5)H3); 2.42 (m, 4 H, CH2CH2CH2CH2SO3); 2.86 (t, 2 H,
CH2CH2COOH, J = 7.5 Hz); 3.44 (t, 2 H, CH2CH2COOH, J =
7.5 Hz); 4.06 (m, 4 H, CH2CH2CH2CH2SO3); 6.31 (d, 2 H,
αꢀCH, α´ꢀCH, J = 13.5 Hz); 6.56 (t, 1 H, γꢀCH, J = 12.0 Hz);
7.17—7.47 (m, 6 H, Ar); 8.26 (m, 2 H, βꢀCH, β´ꢀCH).
5ꢀ(2ꢀCarboxyethyl)ꢀ3,3,3´,3´ꢀtetramethylꢀ1,1´ꢀbis(4ꢀsulfoꢀ
natobutyl)indodicarbocyanine, sodium salt (13). Compound 13
was obtained analogously. The yield was 104 mg (72%),
m.p. 218—219 °C. Found (%): C, 60.04; H, 6.25; N, 3.92.
C
34H42N2O5S. Calculated: M = 590.77. 1H NMR (DMSOꢀd6),
C36H45N2NaO8S2. Calculated (%): C, 59.98; H, 6.29; N, 3.89.
δ: 1.26 (t, 3 H, CH2CH3, J = 7.0 Hz); 1.63 (s, 12 H, 2 C(3)H3,
2 C(3´)H3); 1.73 (m, 4 H, CH2CH2CH2CH2SO3); 2.48 (m,
2 H, CH2CH2CH2CH2SO3); 2.58 (t, 2 H, CH2CH2COOH, J =
7.5 Hz); 2.88 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 4.10 (m,
4 H, CH2CH2CH2CH2SO3, CH2CH3); 6.37 (d, 2 H, αꢀCH,
α´ꢀCH, J = 13.5 Hz); 6.56 (t, 1 H, γꢀCH, J = 12.0 Hz);
7.19—7.63 (m, 7 H, Ar); 8.31 (m, 2 H, βꢀCH, β´ꢀCH).
5ꢀ(2ꢀCarboxyethyl)ꢀ1,1´ꢀdiethylꢀ3,3,3´,3´ꢀtetramethylꢀ5´ꢀ
sulfoindodicarbocyanine (10). A mixture of compound 16b
(53 mg, 0.2 mmol) and malonaldehyde dianil hydrochloride
(17) (59 mg, 0.23 mmol) was heated in acetic anhydride (1.5 mL)
and acetic acid (0.5 mL) at 118 °C for 2 h. Then the solvents
were removed in vacuo. The residue was heated with comꢀ
pound 2a (85 mg, 0.22 mmol) and anhydrous AcOK (120 mg,
1.2 mmol) in acetic anhydride (1.5 mL) and acetic acid (0.5 mL)
at 118 °C for 2 h. Compound 10 was isolated as described
for compound 9. The yield of dye 10 was 60 mg (53%),
m.p. 245—247 °C. Found (%): C, 68.35; H, 6.78; N, 4.95.
C32H38N2O5S. Calculated (%): C, 68.30; H, 6.81; N, 4.98. The
spectroscopic characteristics of the dye are given in Table 1.
MS, m/z: 563.2 [M]+. C32H38N2O5S. Calculated: M = 562.72.
1H NMR (DMSOꢀd6), δ: 1.25 (t, 6 H, CH2CH3, J = 7.0 Hz);
The spectroscopic characteristics of the dye are given in Table 1.
MS, m/z: 699.0 [M]+. C36H45N2O8S2–. Calculated: M = 697.88.
1H NMR (DMSOꢀd6), δ: 1.67 (s, 12 H, 2 C(3)H3, 2 C(3´)H3);
1.75 (m,
8
H, CH2CH2CH2CH2SO3); 2.36 (t,
2
H,
CH2CH2COOH,
J
=
7.5 Hz); 2.43 (m, 4
H,
CH2CH2CH2CH2SO3); 2.85 (t, 2 H, CH2CH2COOH, J =
7.5 Hz); 4.06 (m, 4 H, CH2CH2CH2CH2SO3); 6.33 (d, 2 H,
αꢀCH, α´ꢀCH, J = 13.5 Hz); 6.58 (t, 1 H, γꢀCH, J = 12.0 Hz);
7.19—7.57 (m, 7 H, Ar); 8.28 (m, 2 H, βꢀCH, β´ꢀCH).
5ꢀ(2ꢀCarboxyethyl)ꢀ1´ꢀethylꢀ3,3,3´,3´ꢀtetramethylꢀ5´ꢀ
sulfoꢀ1ꢀ(4ꢀsulfonatobutyl)indodicarbocyanine, sodium salt (14).
Compound 14 was obtained analogously. The yield was 39 mg
(28%), m.p. 230—232 °C. Found (%): C, 58.98; H, 5.92; N, 4.06.
C34H41N2NaO8S2. Calculated (%): C, 58.94; H, 5.96; N, 4.04.
The spectroscopic characteristics of the dye are given in Table 1.
MS, m/z: 670.8 [M]+. C34H41N2O8S2–. Calculated: M = 669.83.
1H NMR (DMSOꢀd6), δ: 1.23 (t, 3 H, CH2CH3, J = 7.0 Hz);
1.67 (s, 12 H, 2 H3C(3), 2 C(3´)H3); 1.76 (m,
4 H,
CH2CH2CH2CH2SO3); 2.25 (t, H, CH2CH2COOH,
2
J = 7.5 Hz); 2.47 (m, 2 H, CH2CH2CH2CH2SO3); 2.83
(t, 2 H, CH2CH2COOH, J = 7.5 Hz); 4.90 (m, 4 H,
CH2CH2CH2CH2SO3, CH2CH3); 6.32 (d, 2 H, αꢀCH, α´ꢀCH,
J = 13.5 Hz); 6.56 (t, 1 H, γꢀCH, J = 12.0 Hz); 7.23—7.62 (m,
6 H, Ar); 8.28 (m, 2 H, βꢀCH, β´ꢀCH).
1.69 (s, 12 H,
2 C(3)H3, 2 C(3´)H3); 2.32 (t, 2 H,
CH2CH2COOH, J = 7.5 Hz); 2.86 (t, 2 H, CH2CH2COOH,

Indodicarbocyanine dyes
Russ.Chem.Bull., Int.Ed., Vol. 56, No. 11, November, 2007 2267
5ꢀ(2ꢀCarboxyethyl)ꢀ3,3,3´,3´ꢀtetramethylꢀ5´ꢀsulfoꢀ1,1´ꢀ
bis(4ꢀsulfonatobutyl)indodicarbocyanine, disodium salt (15).
Compound 15 was obtained as described for compound 10. The
yield was 55 mg (33%), m.p. >250 °C. Found (%): C, 52.59;
H, 5.33; N, 3.42. C36H44N2Na2O11S3. Calculated (%): C, 52.54;
3. A. J. G. Mank, H. T. C. van der Laan, C. Gooijer, U. A. Th.
Brinkman, and N. H. Velthorst, Anal. Chem., 1995, 67, 1742.
4. R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis,
and A. S. Waggoner, Bioconjugate Chem., 1993, 4, 105.
5. A. J. G. Mank, E. J. Molenaar, H. Lingeman, C. Gooijer,
U. A. Th. Brinkman, and N. H. Velthorst, Anal. Chem.,
H, 5.39; N, 3.40. The spectroscopic characteristics of the dye
are given in Table 1. MS, m/z: 777.6 [M]+. C36H44N2O11S3
Calculated: M = 776.94. 1H NMR (DMSOꢀd6), δ: 1.67 (s, 12 H,
2 C(3)H3, 2 C(3´)H3); 1.75 (m, 8 H, CH2CH2CH2CH2SO3);
2.28 (t, 2 H, CH2CH2COOH, J = 7.5 Hz); 2.44 (m, 4 H,
CH2CH2CH2CH2SO3); 2.84 (t, 2 H, CH2CH2COOH, J =
7.5 Hz); 4.09 (m, 4 H, CH2CH2CH2CH2SO3); 6.28 (d, 2 H,
αꢀCH, α´ꢀCH, J = 13.5 Hz); 6.58 (t, 1 H, γꢀCH, J = 12.0 Hz);
7.26—7.75 (m, 6 H, Ar); 8.25 (m, 2 H, βꢀCH, β´ꢀCH).
.
1993, 65, 2197.
2–
6. 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.
7. Y. Lin, R. Weissleder, and C.ꢀH. Tung, Bioconjugate Chem.,
2002, 13, 605.
8. P. L. Southwick, J. G. Cairns, L. A. Ernst, and A. S.
Waggoner, Org. Prep. Proced. Int., 1988, 20, 279.
9. Preparatyka organiczna, Panstwowe Wydawnictwa
Techniczne, Warszawa, 1954 (in Polish).
10. I. M. Hunsberger, E. R. Shaw, J. Fugger, R. Ketcham, and
D. Ledniger, J. Org. Chem., 1956, 21, 394.
This work was financially supported by the Internaꢀ
tional Scientific and Technical Center (Grant 2906).
11. H. Illy and L. Funderburk, J. Org. Chem., 1968, 33, 4283.
12. 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 (Engl. Transl.)].
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Received May 25, 2007;
in revised form August 2, 2007