8
4
JOHANSSON et al.
solution of bistrifluoroacetate of diamine (II) (5 mg, TFA; B); UV (5 mM Me NOH in MeOH): λ 497 nm
4
max
4
+
6
.92 µmol) in 1 : 3 chloroform–isopropanol (2 ml) was
(
ε 7.2 × 10 ); ESI-MS: m/z: 600 [M] .
treated with NBD chloride (14 mg, 70 µmol) in the
Fluorescence measurements were carried out in
same solvent mixture (1 ml) and then with 1 M
NaHCO (150 µl). The reaction mixture was vigorously
quartz cells (10 × 10 mm) at 20°C. Excitation spectra
were registered at the emission wavelengths (520, 550,
and 550 nm) and the emission spectra of fluorescence,
at the excitation wavelengths 340, 470, and 460 nm for
probes (III), (IV) and (VIII, IX), respectively.
3
stirred for 2 h at 55°C in argon atmosphere and then
chromatographed on silica gel using chloroform as an
eluent to yield 4 mg (71%) of (IV) as a chromatograph-
ically homogeneous amorphous red substance; R 0.40
f
on Kieselgel 60 (19 : 1 chloroform–isopropanol, A–C);
ACKNOWLEDGMENTS
We are grateful to Dr. Bo Ek (Swedish Agricultural
University, Uppsala) for recording mass spectra.
4
UV (EtOH): λ
469 nm (ε 4.1 × 10 ); MALDI-MS,
max
+
m/z: 821 [M] .
Dotriacontanedioic acid (VI). A solution of dime-
This work was supported by the Russian Foundation
thyl ester (V) in hot isopropanol (25 ml) was mixed for Basic Research, projects no. 00-04-48416 and 02-04-
with water (4 ml) and 45% NaOH (0.6 ml). The reac- 48287, and Swedish Royal Academy of Sciences.
tion mixture was kept for 12 h at 60°ë and acidified in
a heated state with 3 M HCl to pH 2. The residue was
filtered, washed with water, and dried to yield 56 mg
REFERENCES
. Karolin, J. and Johansson, L.B.-Å., Trends Phys. Chem-
istry, 1997, vol. 6, pp. 171–185.
1
2
3
(
98%) of diacid (VI) (colorless powder); mp 123–
25°ë; 125–127°C (after recrystallization from toluene
with 1% ëç ëééç) (literature data: mp 124–127°C
1
. Wu, P.G. and Brand, L., Anal. Biochem., 1994, vol. 218,
3
pp. 1–13.
[17] and 128–129°C [18]).
. Bergström, F., Hagglof, P., Karolin, J., Ny, T., and
Johansson, L.B.-Å., Proc. Natl. Acad. Sci. USA, 1999,
vol. 96, pp. 12477–12481.
. Kalinin, S.V., Molotkovsky, J.G., and Johansson, L.B.-Å.,
Spectrochim. Acta, Part A, 2002, vol. 58, pp. 1087–
Bis(fluoresceinyl-5-methylamid) of dotriacon-
tanedioic acid (VIII). 1-Methylimidazol (15 µl) and
BOP reagent (4.4 mg, 9.9 µmol) were added to a sus-
pension of (VI) (2.3 mg, 4.5 µmol) in a mixture of
dichloromethane (0.7 ml) and THF (1 ml). The reaction
mixture was stirred for 24 h in argon atmosphere, and
the resulting solution was mixed with N-ethyldiisipro-
pylamine (15 µl) and hydrochloride of 5-(amionome-
thyl)fluorescein (VII) (4.8 mg, 12 µmol), and then
stirred for 6 days with adding THF to offset its loss due
to evaporation. The reaction was monitored by TLC on
Nano-SIL C -100 plates using 39 : 10 : 1 methanol–
4
5
6
7
1
097.
. Grechishnikova, I.V., Johansson, L.B.-Å., and Molot-
kovsky, J.G., Chem. Phys. Lipids, 1996, vol. 81, pp. 87–
9
8.
. Karolin, J., Bogen, S.-T., Johansson, L.B.-Å., and
Molotkovsky, J.G., J. Fluorescence, 1995, vol. 5,
pp. 279–284.
. Johansson, L.B.-Å., Bergström, F., Edman, P., Grechish-
nikova, I.P., and Molotkovsky, J.G., J. Chem. Soc., Fara-
day Trans., 1996, vol. 92, pp. 1563–1567.
1
8
water–TFA system (C). The residue after solvent evap-
oration was dissolved in methanol (2 ml), mixed with
tetramethylammonium hydroxide (~1 mg) and chro-
matographed on a LiChroprep RP-18 column (5 ×
8. Molotkovsky, Yul.G., Bioorg. Khim., 1999, vol. 25,
pp. 855–867.
9
. Thompson, D.H., Wong, K.F., Humphry-Baker, R.,
Wheeler, J.J., Kim, J.-M., and Rananavare, S.B., J. Am.
Chem. Soc., 1992, vol. 114, pp. 9035–9042.
0. Duffin, G.R., Ellames, G.J., Hartmann, S., Herbert, J.M.,
and Smith, D.I., J. Chem. Soc., Perkin Trans. 1, 2000,
pp. 2237–2242.
7
0 mm, 0.9 g) using methanol as eluent to yield 1.2 mg
22%) of pure (VIII); amorphous orange substance; Rf
.40 on Nano-SIL C -100 plates (20 : 50 : 10 : 5
(
1
1
0
1
8
dichloromethane–methanol–water–TFA; B and C;
starting (VII) has R ~ 0.9); UV (5 mM Me NOH in
f
4
1. Waggoner, S.S. and Stryer, L., Proc. Natl. Acad. Sci.
5
MeOH): λ
497 nm (ε 1.4 × 10 ); ESI-MS: m/z:
USA, 1970, vol. 67, pp. 579–589.
max
+
1
197.56 [M] .
12. Chattopadhyay, A., Chem. Phys. Lipids, 1990, vol. 53,
pp. 1–15.
5
-(Hexadecanoylaminomethyl)fluorescein (IX).
1
3. Haugland, R.P., Handbook of Fluorescent Probes and
Research Chemicals, 6th ed., Spence, M.T.Z., Ed.,
Eugene, OR: Molecular Probes, 1996, pp. 19–22.
4. Lin, S. and Struve, W.S., Photochem. Photobiol., 1991,
vol. 54, pp. 361–365.
A suspension of 5-(aminomethyl)fluorescein hydro-
chloride (VII) (2 mg, 5 µmol) in a mixture of THF
1 ml), water (0.1 ml), and 1-methylimidazol (20 µl)
(
1
1
was stirred for 5 min and treated with a 10% solution of
palmitoyl chloride (36 µmol) in THF (100 µl). The
reaction mixture was stirred for 2 h and then evaporated
in a vacuum. A chromatography of the residue on
LiChroprep RP-18 [as described in the synthesis of
5. Sjöback, R., Nygren, J., and Kubista, M., Spectrochim.
Acta, Part A, 1995, vol. 51, pp. 7–21.
1
1
6. German Patent (D.R.P.), 701953, 1935.
7. Buchta, E. and Huhn, C., Liebigs Ann. Chem., 1966,
vol. 695, pp. 42–48.
(
VIII)] yielded 2.4 mg (80%) of (IX) as amorphous
orange substance; R 0.5 on Nano-SIL C -100 plates
f
18
18. Hünig, S. and Buysch, H.-J., Chem. Ber., 1967, vol. 100,
(20 : 50 : 10 : 5 dichloromethane–methanol–water–
pp. 4017–4026.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003