Synthesis of new photoaffine probes on the basis of ganglioside GM1

Article abstract of DOI:10.1023/A:1015025609230

New photoaffine probes, photoreactive derivatives of ganglioside GM1 bearing a carbene-generating diazocyclopentadien-2-ylcarbonyl group at various distances from the carbohydrate moiety in their molecules were synthesized.

Full text of DOI:10.1023/A:1015025609230

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 2, 2002, pp. 152–157. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 2, 2002, pp. 173–179.
Original Russian Text Copyright © 2002 by Tsibizova, Vodovozova, Mikhalyov, Molotkovsky.
Synthesis of New Photoaffine Probes on the Basis
of Ganglioside GM1
1
E. V. Tsibizova, E. L. Vodovozova, I. I. Mikhalyov, and Yul. G. Molotkovsky
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, GSP-7 Moscow, 117997 Russia
Received June 13, 2001; in final form, July 02, 2001
Abstract—New photoaffine probes, photoreactive derivatives of ganglioside GM1 bearing a carbene-generat-
ing diazocyclopentadien-2-ylcarbonyl group at various distances from the carbohydrate moiety in their mole-
cules were synthesized.
Key words: diazocyclopentadien-2-ylcarbonyl label, gangliosides, lipid derivatives, photoaffinity labeling
1
INTRODUCTION
ture of its oligosaccharide determinant and, therefore,
allows the preparation of lipophilic probes for the study
of various lipid–protein interactions inside the mem-
branes.
Gangliosides, glycosphingolipids bearing one or
several residues of sialic acid, are the obligatory com-
ponents of eukaryotic plasmatic membranes. Espe-
2
Whereas phospholipid probes have long been used
in membrane research, the use of ganglioside probes is
rare. This is likely due to their difficult synthesis. As the
total synthesis of labeled gangliosides is very laborious
and unpractical, it is more appropriate to modify natu-
ral lipids at both their oligosaccharide (see, e.g., [18])
and ceramide moieties (see, e.g., [19–21]). The most
effectual route for modification of the second type is the
replacement of a fatty acid residue in a natural ganglio-
side molecule by a labeled acyl residue. This opportu-
nity has appeared due to the development of the meth-
ods for the removal of acyl residue from the ceramide
moiety of gangliosides by alkaline hydrolysis [22].
A number of groups (e.g., aryl azide, diazirine, and
benzophenone) were recommended as photoreactive
(photoaffinity, photoactivatable) labels, because they
form highly reactive intermediates (nitrenes, carbenes,
or biradicals) upon irradiation. However, none of them
fully meets all the requirements imposed on such
groups.
A relatively easy way for the synthesis of aryl azide
derivatives is undoubtedly an important advantage for
their practical use. Therefore, nitrene-generating phe-
nyl azide groups are those most often used for PAL
(see, e.g., [23]), and their photochemistry has been
rather comprehensively studied. Phenyl azides can be
photolyzed under rather mild conditions, which
decrease the probability of damage to biomolecules
(λ > 300 nm; though their absorption maxima are at
λ < 300 nm). However, nitrenes resulting from their
photolysis are low-reactive, which explains a low cross-
linkage level of the probes to biomolecules.
cially frequent in nervous tissue, they play an important
role in intercellular interactions and in cell interactions
with various exogenous factors [1]. Gangliosides are
predominantly located on the outer side of the cell
membrane bilayer where oligosaccharide determinants
are exposed on the cell surface [1, 2]. It is known that
they serve as receptors for various toxins [3–5], bacte-
ria [6], and viruses [7, 8]; participate in the recognition
processes by changing the expression of protein recep-
tors [9, 10], modulate protein phosphorylation [11], are
involved in the reception of interleukin-2 and -4 [12,
13], are specific markers of tumor cells [14, 15], and
can suppress immunity [16, 17]. The polar (oligosac-
charide) part of the ganglioside molecule plays the
deciding role in these interactions; however, entire
complexes of protein receptors in proximity to the apo-
lar (ceramide) part of the molecule inside the cell mem-
brane undergo conformational changes. Moreover, a
direct contact of the ceramide residue with the recepted
protein is also possible; this could result in the cross-
linkage of a photolabeled ganglioside and the protein
ligand.
Photolabeled gangliosides can be valuable tools in
studying the membrane rearrangements that are caused
by a ligand binding to a receptor; in these cases, the
recepted protein directly binds to ganglioside or serves
as a cofactor. It is important that PAL in the apolar part
of the ganglioside molecule does not distort the struc-
1
Please send correspondence to: phone: +7 (095) 330-6610;
e-mail: parfin@mail.ru.
2
Abbreviations: Dcp, diazocyclopentadien-2-ylcarbonyl; DcpOH,
diazocyclopentadien-2-ylcarboxylic acid; lyso-GM1, lysogangli-
Nitrenes generated from aryl azides have also been
found [24] to undergo a rapid rearrangement to long-
oside GM1; Np, p-nitrophenyl; PAL, photoaffinity labeling; Py,
pyridine; and Tf O, trifluoromethanesulfonic anhydride.
2
1068-1620/02/2802-0152$27.00 © 2002 MAIK “Nauka /Interperiodica”

SYNTHESIS OF NEW PHOTOAFFINE PROBES
153
λ > 300 nm
cyclohexane
..
O
O
O
N2
Scheme 1.
living dehydroazepines, which first and foremost react and is stable in the absence of UV light under various
with nucleophiles, such as amino and thiol residues.
conditions: mild basic and acidic media (including per-
acetic acid) [34], heating (at ≥150°C), and oxidizers.
We had previously synthesized the Dcp derivatives of
14C/3H-labeled phosphatidylcholine and sphingomye-
lin [33] and successfully used them for studying the
membrane topography of cytochrome P-450 [35]. We
further investigated some other characteristics of the
Dcp group and found that it can be easily iodinated
under oxidizing conditions, with its ability to generate
a highly reactive carbene upon photoactivation being
retained [36].
Photolysis of diazirine derivatives results in car-
benes, which possess a higher reactivity than nitrenes
[25]. Previously, a number of diazirine derivatives have
been obtained, with 3-(trifluoromethyl)-3-phenyldiaz-
irine group possessing the best characteristics [26]. The
multistage synthesis of diazirine ring hinders the wide
use of these compounds.
In recent years, benzophenone groups have often
been used for PAL. The compounds containing them
have several obvious advantages: they are more chemi-
cally stable than aryl azides and diazirines, are acti-
vated at 350–360 nm, and, after photoactivation, can
react with nonactivated C–H bonds even in the presence
of aqueous solvents [27]. However, this label is very
bulky, and the generated biradical has a certain ste-
reospecificity. Moreover, the benzophenone chro-
mophore needs a durable irradiation for its photoactiva-
tion [28], which is undesirable in some cases.
In this work, we report the synthesis of new photo-
affine probes, ganglioside GM1 derivatives bearing
Dcp group at various distances from the carbohydrate
moiety of the molecule.
RESULTS AND DISCUSSION
1. Long-Chain Dcp-GM1 probe (IX)
To ensure the high sensitivity of a photoaffine probe,
it should include radionuclides with high radiation
When studying biological systems with photoaffin-
ity probes, the presence of a labile bond between the
photoaffinity label and the remainder of the probe mol-
ecule substantially facilitates the analysis of the cross-
linkage products. Study of the lipid–protein interac-
tions is primarily interesting, and, hence, ester bond,
which, unlike peptide bond, is labile under alkaline
conditions, is appropriate as such a labile bond. There-
fore, we synthesized an aliphatic acid bearing Dcp res-
idue on the terminal carbon atom (V) as a synthon for
obtaining various lipid probes, which can be applied in
examining the central region of lipid bilayer in the bio-
logical and model membranes (see Scheme 2).
32
energy (125I, 131I, P, etc.). The lifetime of such com-
pounds is very short; thus, it is reasonable to introduce
the radioisotope into the cold probe immediately before
use. To facilitate further analysis of the cross linkage
products, the direct radiolabeling of the photoaffinity
group is also desirable. Some functionalization meth-
ods for 125I introduction were developed for a number
of photoaffinity labels, namely, perfluorophenyl azide
[29], 3-(trifluoromethyl)-3-phenyldiazirine, [30], and
benzophenone groups [31]. However, this immediate
introduction substantially complicates the probe syn-
thesis.
11-Hydroxyundecanoic acid (I) was synthesized
from the available 11-aminoundecanoic acid by deam-
ination with nitrous acid at heating in water [37]. The
resulting mixture of three products included (according
to TLC) undecylenic acid (Rf 0.45, somewhat less than
a half of the total amount), 10-hydroxyundecanoic acid
(Rf 0.35, negligible amount), and the target product (I)
(Rf 0.2, approximately a half of the total amount), iso-
lated by chromatography on silica gel in 38% yield. Its
1H NMR spectrum contained a triplet resonance char-
acteristic of protons of a methylene group at the pri-
mary hydroxyl (see the Experimental section). Its IR
spectrum also confirmed the structure of the resulting
product (I).
Therefore, a search for new photoaffinity groups
still remains a topical problem in membranology.
The carbene-generating Dcp group was first sug-
gested for PAL by Nielsen et al. [32]. This labeling
group is small in size, a substantial absorption in the
near UV region (λmax 314 nm, ε 15900 å–1 cm–1) is
characteristic of it, and it is readily photolyzed to give
carbene. The carbene was shown to be highly reactive:
it is effectively inserted into nonactivated C–H bonds
3
[33] (Scheme 1). Starting DcpOH can be easily
obtained by three-stage synthesis from cyclopentadiene
3
1
The structural study of the insertion product by H NMR will be
published elsewhere.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 2 2002

154
TSIBIZOVA et al.
NH2(CH2)10COOH
a
h
(I) HO(CH2)10COOH
DcpO(CH2)10COONp (VI)
(IX)
b
g
(II) HO(CH2)10COOMe
DcpO(CH2)10COOH (V)
c
e, f
d
d
(III) TfO(CH2)10COOMe
DcpO(CH2)10COOMe (IV)
DcpOCH2COONp (VIII)
h
(VII) ICH2COONp
(X)
i
ICH2COOH
OH
( )n
Galβ1-3GalNAcβ1-4Galβ1-4GlcβO
n = 8 (~60%)
n = 10 (~40%)
3
NH2
NeuNAcα2
CO(CH )
OOC
2
q
N2
Dcp
(IX), q = 10 (yield 98%)
(X), q = 1 (yield 88%)
Scheme 2.
i
Reagents: a. HNO ; b. MeOH/HCl; c. Tf O/Py; d. DcpOH/Et N; e. KOH, Pr OH/H O; f. HCl; g. CF COONp/Py; h. lyso-
2
2
3
2
3
GM1/Et N; i. HONp, DCC.
3
Methyl 11-hydroxyundecanoate (II) was obtained
The long-chain ganglioside probe (IX) was synthe-
by the esterification of (I) with a methanolic HCl in sized by the reaction of lyso-GM1 with p-nitrophenyl
53% yield. Its treatment with Tf2O in the presence of ester (VI) in DMSO in the presence of triethylamine
and isolated by gel filtration in yield (98%), determined
from its mass and UV-absorption of the Dcp label.
pyridine in chloroform at –10°ë led to methyl 11-(tri-
fluoromethanesulfonyloxy)undecanoate (III) (yield
86%), which was further used without additional puri-
fication.
2. Short-Chain Dcp-GM1 probe (X)
The reaction of triflate (III) with DcpOH in anhy-
drous acetone in the presence of a small excess of tri-
ethylamine gave methyl 11-(Dcp-oxy)undecanoate
(IV) in 50% yield. The structure of (IV) was confirmed
by mass spectrometry and 1H NMR spectroscopy.
To obtain probe (X) bearing the label in the close
proximity to the ganglioside polar head, we somewhat
changed the synthetic scheme.
We failed to obtain p-nitrophenyl iodoacetate (VII)
in the same way as ester (VI) using p-nitrophenyl tri-
fluoroacetate, because the reagent had almost the same
chromatographic mobility as the target product. Ester
(VII) was obtained by the esterification of iodoacetic
acid with p-nitrophenol in the presence of DCC. The
structure of the resulting compound was confirmed by
1H NMR and elemental analysis.
Methyl ester (IV) was hydrolyzed with KOH in
aqueous isopropanol at room temperature. The rate of
alkaline hydrolysis of the ester bond formed by DcpOH
is substantially lower than that of the aliphatic ester.
The reaction was carried out for 20 h until the appear-
ance of traces of free DcpOH (TLC). The successive
chromatography on silica gel and the reversed phase
DcpOH was treated with (VII) in anhydrous ace-
yielded 40% of 11-(Dcp-oxy)undecanoic acid (V), tone in the presence of triethylamine. The reaction
which was then transformed into its p-nitrophenyl ester product, Dcp-oxyacetic acid p-nitrophenyl ester (VIII),
(VI) with p-nitrophenyltrifluoroacetate in pyridine.
was isolated by column chromatography on silica gel in
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SYNTHESIS OF NEW PHOTOAFFINE PROBES
155
84% yield, and its structure was confirmed by mass plasma desorption time-of-flight mass spectrometer
spectrometry and 1H NMR.
(ionization with the products of 252Cf fission at an
accelerating voltage of +15 eV) or on a Finnigan MAT
9005 (ESI) mass spectrometer. IR spectra were
recorded on a Specord 75IR (Carl Zeiss, Germany)
instrument. H NMR spectra were registered on a
Bruker WM-500 (United States) spectrometer.
Photoreactive ganglioside (X) bearing the Dcp-oxy-
acetyl residue was synthesized, like (IX), from lyso-
GM1 and Dcp-oxyacetic acid p-nitrophenyl ester (VIII).
Its yield (88%) was somewhat less than that of (IX),
which is obviously due to the steric hindrances during
acylation of the amino group of sphingosine residue.
High yields of probes (IX) and (X) can be explained
by the advantages of our approach, namely, the use of
DMSO as a solvent for the reaction of lysoganglioside
with active esters (VI) and (VIII) and the use of gel fil-
tration instead of absorption and reversed phase chro-
matography for the purification of micromolar amounts
of target products.
1
11-Hydroxyundecanoic acid (I). A solution of
NaNO2 (483 mg, 7 mmol) in water (2 ml) was added to
a solution of 11-aminoundecanoic acid (1 g, 5 mmol) in
a 4 : 1 water–acetic acid mixture (5 ml). The reaction
mixture was heated for 1 h in a boiling water bath
(100°ë), kept overnight at room temperature, acidified
with 1 N HCl to pH 3.0, and extracted with chloroform
(3 × 5 ml). The extract was filtered though cotton wool,
evaporated, dried, and chromatographed on Silica gel L
(100–160 µm). Elution with an 0
10% gradient of
EXPERIMENTAL
10 : 1 isopropanol—acetic acid in chloroform gave
382 g (38%) of (I) as a white solid, Rf 0.2 (A; a and b).
The product (white needles from water) had: mp 64–
65°ë (lit. [39]: mp 65–66°ë); IR (ν, cm–1): 3620 (OH),
2933 and 2860 (ëç2), 1700 (C=O), and 1226 and 1200
11-Aminoundecanoic
acid,
trifluoromethane-
sulfonic anhydride, trifluoroacetic anhydride, and tri-
ethylamine were from Fluka (Switzerland); DMSO and
anhydrous pyridine from Merck (Germany); and
iodoacetic and acetic acids (reagent grade) were of
domestic production. DCC (Serva, Germany) was used
as 1 M solution in tetrachloromethane. To remove
water, acetone was distilled over K2CO3 and chloro-
form, over P2O5. Other reagents and solvents (Reakhim,
Russia) were purified by standard procedures. p-Nitro-
phenyl trifluoroacetate was obtained from p-nitrophenol
and trifluoroacetic anhydride [38]. DcpOH was synthe-
sized as described in [34]. Lysoganglioside was obtained
from the natural ganglioside by hydrolysis [21].
1
(OH–COOH); H NMR (CDCl3, δ, ppm): 1.29 (12 H,
m, (ëç2)6), 1.55 (2 H, m, ëç2ëç2ëééç); 1.64 (2 H,
m, çéëç2ëç2), 2.35 (2 H, t, ëç2ëééç), and 3.64
(2 H, t, çéëç2).
Methyl 11-hydroxyundecanoate (II). Acetyl chlo-
ride (5 ml) was added dropwise to methanol (50 ml) at
vigorous stirring, and the resulting mixture was imme-
diately added to acid (I) (200 mg, 1 mmol). The reac-
tion mixture was gently stirred for 3.5 h at 40°ë and
evaporated. The residue was chromatographed on Sil-
ica gel L (40–100 µm); elution with system B gave
115 mg (53%) of ester (II) as a colorless oil; Rf 0.45 (A; a).
Methyl 11-(trifluoromethanesulfonyloxy)unde-
canoate (III). Ester (II) (115 mg, 0.53 mmol) was dis-
solved in anhydrous chloroform (6 ml) containing
anhydrous Py (70 µl, 0.87 mmol) and the resulting
solution was added dropwise to a solution of Tf2O
(144 µl, 0.87 mmol) in anhydrous chloroform (6 ml)
under stirring and cooling in an ice–salt bath (–10°ë).
The mixture was then stirred for 2 h in the stream of
argon at 0°ë and then extracted with water (3 × 3 ml).
The organic phase was filtered through cotton wool, con-
centrated, and dried to give 159 mg (86%) of ester (III)
as a chromatographically pure colorless oil; Rf 0.8 (A; a).
Column chromatography was carried out on Silica
gel 60 (40–63 µm; Merck, Germany) and Silica gel L
(40–100 and 100–160 µm; Chemapol, Czech Repub-
lic). Silica gel RP-18 (25–40 µm; Merck, Germany)
was used for the reversed phase chromatography. Gel
filtration was carried out on Sephadex LH-20 (Pharma-
cia, Sweden). For TLC, the silica gel 60 F254 and RP-18
F254 (Merck, Germany) precoated plates were used; sol-
vent systems were: (A) 50 : 10 : 1 toluene–ethyl ace-
tate–acetic acid, (B) 9 : 1 chloroform–methanol, (C) 7 :
3 methanol–water, (D) 9 : 1 methanol–water, (E) 1 : 1
methanol–water, (F) 6 : 4 methanol–water, (G) 1 : 1
chloroform–methanol, and (H) 2 : 3 : 1 isopropanol–
ethyl acetate–water. Substance spots were visualized
by (a) 5% phosphomolybdic acid in ethanol, (b) water,
(c) UV irradiation, (d) ammonia vapor, (e) 0.2% ninhy-
drin in ethanol, and (f) resorcinol. Solvents were evap-
orated in a vacuum at temperatures below 40°ë. After
purification, the reaction products were dried at 20 Pa.
Melting points were determined on a Kofler apparatus
and were uncorrected.
All manipulations with Dcp derivatives were carried
out under yellow light.
UV spectra were registered on an Ultrospec II 4050
(LKB, Sweden) spectrophotometer in ethanol. Mass
spectra were taken on a MSBKh (Sumy, Ukraine)
Methyl 11-(Dcp-oxy)undecanoate (IV). Ester
(III) (159 mg, 0.46 mmol) in anhydrous acetone
(2.6 ml) was added to a solution of DcpOH (20 mg,
0.15 mmol) and triethylamine (80 µl, 0.58 mmol) in
anhydrous acetone (1 ml). The reaction mixture was
stirred for 40 min at room temperature and evaporated.
The residue was chromatographed on a Silica gel 60
column eluted with a 0
7% gradient of ethyl ace-
tate in heptane to give 25 mg (50%) of (IV) as a chro-
matographically pure yellowish oil; Rf 0.63 (A; a and
c); MS (252Cf), m/z: 334 [M]+; UV [λmax, nm (ε)]: 215
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 2 2002

156
TSIBIZOVA et al.
(12 400) and 314 (15 900); 1H NMR (CDCl3, δ, ppm):
1.29 (12 H, m, (ëç2)6), 1.38 (2 H, m), 1.61 (2 H, m),
1.70 (2 H, t, J 6.72 Hz, ëç2ëéé), 3.66 (3 H, s, ëç3),
4.24 (2 H, t, Dcpéëç2), 6.07 (1 H, dd, J4'5' 4.60 Hz and
J4'3' 3.20 Hz, H4'), 6.82 (1 H, dd, J3'4' 3.18 Hz and J3'5'
1.96 Hz, H3'), and 7.02 (1 H, dd, J5'4' 4.64 Hz and J5'3'
1.96 Hz, H5').
(28 µl, 200 µmol) in anhydrous acetone (1 ml) was
stirred for 2–3 min, and ester (VII) (31.5 mg,
0.1 mmol) in anhydrous acetone (1 ml) was added. The
solution was stirred overnight and evaporated. The res-
idue was chromatographed on a reversed phase column
successively eluted with systems E and F to yield 25 mg
(80%) of chromatographically pure (VIII); Rf 0.67 (A;
c and d); MS (252Cf), m/z: 315.7 [M]+; 1H NMR (CDCl3,
δ, ppm): 5.04 (2 H, s, éëç2ëéé), 6.14 (1 H, dd, J4'5'
4.58 Hz, J4'3' 3.30 Hz, H4'), 6.97 (1 H, dd, J3'4' 3.10 Hz,
J3'5' 2.14 Hz, H3'), 7.12 (1 H, dd, J5'4' 4.58 Hz, J5'3'
1.98 Hz, H5'), 7.36 (2 H, d, J2'3' = J6'5' = 9.15 Hz, H2'
and H6'), and 8.30 (2 H, d, J3'2' = J5'6' = 9.15 Hz, H3'
and H5'). A solution of ester (VIII) in DMSO
(0.38 µmol/50 µl) was prepared.
11-(Dcp-oxy)undecanoic acid (V). A solution of
ester (V) (25 mg, 75 µmol) in isopropanol (16 ml) and
5% KOH (0.4 ml) was kept until the appearance of
traces of DcpOH (TLC monitoring; system A; a and c).
The reaction was stopped by neutralization with 1 N
HCl up to pH 7.0, evaporated, and the dry residue was
suspended in water (1 ml), acidified up to pH 2.0–3.0,
and extracted with chloroform (3 × 0.5 ml). The extract
was washed with a small amount of water and filtered
through cotton wool. The filtrate was evaporated and
chromatographed on a Silica gel 60 column eluted with
11-(Dcp-oxy)undecanoyl GM1 (IX). Triethyl-
amine (1 µl, 7 µmol) and a solution of (VI) (530 µg,
1.2 µmol) in DMSO (50 µl) were added to lyso-GM1 (1
mg, ~1 µmol) in DMSO (50 µl). The reaction mixture
was kept for 16 h at room temperature, treated with a
drop of water, diluted with system F (0.1 ml), and
applied onto a Sephadex LH-20 column (0.5 × 20 cm)
equilibrated with system F. Elution with system E
resulted in (IX); yield 98% (from UV absorption); Rf
a 0
5% gradient of 10 : 1 isopropanol–acetic acid
in chloroform. Fractions containing (V) and traces of
DcpOH were combined and chromatographed on a
reversed phase column successively eluted with sys-
tems C and D to yield 8.2 mg (34%) of acid (V) as a yel-
lowish amorphous substance; Rf 0.44 (A; a and c); its
UV spectrum was similar to that of ester (IV).
0.25, Rf start 0.07 (E; a, c, e, and f); 1H NMR (CD3OD,
δ, ppm): 1.08 (3 H, t, J 7.10 Hz, CH3), 1.45–1.55 (~36
H, m), 1.91 (2 H, m, CH2CH2CO); 2.11 (1 H, dd ~ t, J
12.10 Hz, H3a of Neu5Acα), 2.20 and 2.18 (2 × 3 H, 2
s, 2 NAc), 2.36 (2 H, t, J 7.52 Hz, CH2CO), 2.92 (1 H,
dd, 2J 12.20 Hz, 3J 4.42 Hz, H3e of Neu5Acα), 4.05 (1
H, m, H2), 4.63, 4.55, and 4.49 (3 × 1 H, 3 dd, J 7.61,
8.03, and 7.81 Hz, 3 H1 of hexapyranoses β), 5.64 (1 H,
dd, J4'5' 15.21, J4'3' 7.78 Hz, H4 of (E)C=C), 5.87 (1 H,
dt, J5'4' 15.34, J5'6' 7.72 Hz, H5 of (E)C=C), 6.26 (1 H, br.
dd, J4'5' 4.37 and J4'3' 3.46 Hz, H4'), 6.97 (1 H, br. t (dd),
J3'4' 3.40 and J3'5' 1.80 Hz, H3'), and 7.35 (1 H, br. dd,
J5'4' 4.58 and J5'3' 1.83 Hz, H5'); MS (ESI), m/z:
1582.875 [M1 + H]+, 1604.869 [M1 + Na]+, 1620.837
[M1 + K]+ {calculated for C72H119N5O33 [M1]+ =
1581.7885 and [M1 + H]+ = 1582.7963}; 1610.912
[M2 + H]+, 1632.893 [M2 + Na]+, 1648.866 [M2 + K]+
{calculated for C74H123N5O33 [M2]+ = [M1]+ + 2CH2 =
1609.8198, [M2 + H]+ = 1610.8276); UV spectrum is
similar to that of ester (VI).
p-Nitrophenyl 11-(Dcp-oxy)undecanoate (VI).
p-Nitrophenyl trifluoroacetate (5.8 mg, 0.024 mmol) in
anhydrous Py (200 µl) was kept with acid (V) (4 mg,
12 µmol) at room temperature for 5 h under gentle stir-
ring. The reaction mixture was evaporated and chro-
matographed on a Silica gel 60 column eluted with a
0
2% gradient of ethyl acetate in toluene to give
2.7 mg (50%) of (VI) as a yellow oil; Rf 0.78 (A; a, c,
and d); MS (252Cf), m/z: 441.6 [M]+. A solution of ester
(VI) in DMSO (1 µmol/50 µl) was prepared.
p-Nitrophenyl iodoacetate (VII). A solution of
p-nitrophenol (46 mg, 0.36 mmol) and DCC (80 mg,
0.4 mmol) in anhydrous chloroform (3 ml) was cooled
in the ice bath to 0°ë and added to a solution of
iodoacetic acid (56 mg, 0.3 mmol) in anhydrous chlo-
roform (3 ml) under stirring. The mixture was kept for
1 h at 0°ë and overnight at room temperature, filtered
through cotton wool, and evaporated. The dry residue
containing (VII) and dicyclohexylurea was treated with
toluene and filtered. The filtrate was chromatographed
on a Silica gel 60 column eluted with toluene contain-
ing 0.5% acetic acid to give 51 mg (55%) of chromato-
graphically pure (VII); Rf 0.8 (A; c and d). The product
(Dcp-oxy)acetyl GM1 (X) was synthesized as
described for (IX) from lyso-GM1 (~1 µmol) and
was crystallized from ethanol to give white monoclinic (VIII) (380 µg, 1.2 µmol); yield 88% (from UV
1
1
crystals; mp 80°ë; H NMR (CDCl3, δ, ppm): 3.95 absorption); Rf 0.15 (E; a, c, e, and f); H NMR
(2H, s, Iëç2CO), 7.33 (2 H, d, J2'3' = J6'5' 9.17 Hz, 2'-H (CD3OD, δ, ppm): 1.09 (3 H, t, J 7.10 Hz, CH3), 1.45–
and 6'-H), and 8.30 (2 H, d, J3'2' = J5'6' = 9.17 Hz, H3' 1.57 (~22 H, m), 2.20 (2 × 3 H, br. s, 2 NAc), 2.85 (2 H,
2
3
and H5'). Found, %: C, 31.24; H, 2.00; N, 4.42; s, ëéCH2ODcp), 2.92 (1 H, dd, J 12.20 and J
I, 41.00. C8H6INO4. Calculated, %: C, 31.29; H, 1.97;
N, 4.56; I, 41.33.
p-Nitrophenyl Dcp-oxyacetate (VIII). A solution
of DcpOH (13.8 mg, 0.1 mmol) and triethylamine
4.40 Hz, H3e of Neu5Acα), 4.03 (1 H, m, H2), 4.63,
4.59, and 4.50 (3 × 1 H, 3 dd, J 7.60, 8.02, and 8.02 Hz,
3 H1 of hexapyranoses β), 5.65 (1 H, dd, J4'5' 15.20 and
J4'3' 7.72 Hz, H4 of (E)C=C), 5.89 (1 H, dt, J5'4' 15.32,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 2 2002

SYNTHESIS OF NEW PHOTOAFFINE PROBES
157
14. Fredman, P., Prog. Brain Res., 1994, vol. 101, pp. 225–
J5'6' 7.70 Hz, H5 of (E)C=C), 6.29 (1 H, br. t (dd), J4'5'
4.30 and J4'3' 3.30 Hz, H4'), 7.09 (1 H, br. t, H3'), and
7.40 (1 H, br. dd, J5'4' 4.60 and J5'3' 1.80 Hz, H5'); MS
(ESI), m/z: 1456.740 [M1 + H]+, 1478.714 [M1 + Na]+,
1494.701 [M1 + K]+ {calculated for C63H101N5O33
[M1]+ = 1455.6477 and [M1 + H]+ = 1456.6555},
1484.723 [M2 + H]+, 1506.740 [M2 + Na]+, 1522.722
[M2 + K]+ {calculated for C65H105N5O33 [M2]+ = [M1]+ +
2CH2 = 1483.6790 and [M2 + H]+ = 1484.6868}. UV
spectrum is similar to that of ester (VI).
240.
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ACKNOWLEDGMENTS
We are grateful to Dr. A.B. Tuzikov (Shemyakin–
Ovchinnikov Institute of Bioorganic Chemistry, Rus-
sian Academy of Sciences) for assistance in interpreta-
tion of 1H NMR spectra and to Dr. Bo Ek (The Swedish
University of Agricultural Sciences, Uppsala) for regis-
tration of high-resolution mass spectra.
1H NMR spectra were registered with the partial
support of the Ministry of Science of the Russian Fed-
eration, project no. 96-03-08, and the Russian Founda-
tion for Basic Research, project no. 00-04-55024.
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