The synthesis of 11-[(2-pyridyl)amino]- and 11-[(9-anthracenylcarbonyl) amino]undecyl phosphate and the study of their acceptor properties in the enzymatic reaction catalyzed by Salmonella galactosyl phosphotransferases

Article abstract of DOI:10.1134/S1068162014010038

Undecyl phosphate derivatives with new fluorescent labels, 11-[(2-pyridyl)amino]undecyl phosphate and 11-[(9-anthracenylcarbonyl)amino] undecyl phosphate, were synthesized. These compounds were shown to be acceptor substrates of the galactosyl phosphate r

Full text of DOI:10.1134/S1068162014010038

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 1, pp. 89–96. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © L.L. Danilov, N.M. Balagurova, A.N. Vinnikova, N.S. Utkina, V.I. Torgov, N.A. Kalinchuk, T.N. Druzhinina, V.V. Veselovsky, 2014, published in Bioorꢀ
ganicheskaya Khimiya, 2014, Vol. 40, No. 1, pp. 99–107.
The Synthesis of 11ꢀ[(2ꢀPyridyl)amino]ꢀ and
11ꢀ[(9ꢀAnthracenylcarbonyl)amino]undecyl Phosphate
and the Study of Their Acceptor Properties in the Enzymatic
Reaction Catalyzed by Salmonella Galactosyl Phosphotransferases
L. L. Danilov, N. M. Balagurova, A. N. Vinnikova, N. S. Utkina1, V. I. Torgov,
N. A. Kalinchuk, T. N. Druzhinina, and V. V. Veselovsky
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
Received July 23, 2013; in final form, September 4, 2013
Abstract—Undecyl phosphate derivatives with new fluorescent labels, 11ꢀ[(2ꢀpyridyl)amino]undecyl phosꢀ
phate and 11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl phosphate, were synthesized. These compounds were
shown to be acceptor substrates of the galactosyl phosphate residue in the enzymatic reaction catalyzed by
galactosyl phosphotransferase from Salmonella anatum or Salmonella newport membrane preparations.
Keywords: Oꢀantigen biosynthesis, 11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl phosphate, galactosyl phosphoꢀ
transferase, 11ꢀ[(2ꢀpyridyl)amino]undecyl phosphate, Salmonella anatum, Salmonella newport
DOI: 10.1134/S1068162014010038
1
INTRODUCTION
transferases [4, 5]. With the goal of increasing the
detection sensitivity of nonisoprenoid substrates, we
synthesized the first members of this series bearing fluꢀ
It was earlier shown in biochemical studies with
E. coli glycosyl transferases that the natural acceptor
substrate (undecaprenyl diphosphate 2ꢀacetamidoꢀ2ꢀ
orescent labels at the
cal: 11ꢀ[(9ꢀanthracenyl)methoxy]undecyl phosphate
and
1ꢀ{11ꢀ[(9ꢀanthracenyl)methoxy]undecyl}ꢀ 2ꢀ
ꢀDꢀgalactopyranosyl) diphosphate [6]. The first
ω
ꢀcarbon atom of the alkyl radiꢀ
deoxyꢀ ꢀDꢀglucopyranose) can be replaced by a synꢀ
α
P
P
thetic derivative of the nonisoprenoid structure conꢀ
taining a phenoxyundecyl group in place of an undeꢀ
caprenyl residue [1]. The phenoxy group allowed the
detection of the compound using its specific UV
absorption (λmax 260–280 nm) in the enzymatic reacꢀ
tions of the assembly of Oꢀantigen polysaccharides.
Biochemical reactions of the oligosaccharide chain
elongation passing by the initiation stage, the transfer
of the corresponding glycosyl phosphate residue to 11ꢀ
phenoxyundecyl phosphate, were studied. We develꢀ
oped a simple method of the synthesis of 11ꢀphenoxꢀ
yundecyl phosphate and first demonstrated the possiꢀ
bility of its use as an acceptor substrate of bacterial glyꢀ
cosyl phosphotransferases [2, 3]. Later, we synthesized
11ꢀphenoxyundecyl diphosphate sugars including
previously unpublished ones and used them in bioꢀ
chemical reactions of assembling Salmonella and
Escherichia coli Oꢀantigen repeat units catalyzed by
the proper glycosyl phosphotransferases and glycosyl
(
α
compound was shown to be an acceptor substrate of
the galactosyl phosphate residue, and the second one,
of the mannose residue in enzymatic reactions cataꢀ
lyzed by galactosyl phosphotransferase and mannosyl
transferase respectively from S. newport membrane
preparations. We also synthesized 2ꢀacetamidoꢀ2ꢀ
deoxy sugar undecyl diphosphate derivatives and
showed their potential as acceptor substrates of bacteꢀ
rial glycosyl transferases [7–9]. The introduction of a
fluorophore group enabled an increase in the sensitivꢀ
ity of product detection in biochemical reactions if
compared with similar compounds containing a pheꢀ
noxy group and made the detection level comparable
with that for the radiolabelled derivatives.
This work is a result of our ongoing studies. We synꢀ
thesized two new undecyl phosphate derivatives,
11ꢀ[(2ꢀpyridyl)amino]undecyl phosphate (VIII) and
11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl
phosꢀ
phate (XVI), in which the fluorophore groups are
linked to alkyl radicals by an amino or imido group
respectively (in contrast to the earlier synthesized
compounds of this series that have an ether bond in
this position). The capacity of these compounds to
Abbreviations: DBU, 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene;
DHP, 3,4ꢀdihydroꢀ2Hꢀpyran; PCC, pyridinium chlorochroꢀ
mate; PPTS, pyridinium
dropyranyl.
Corresponding author: phone: +7 (499) 137ꢀ75ꢀ70; eꢀmail:
utkinans@yahoo.com.
pꢀtoluenesulfonate; Thp, 2ꢀtetrahyꢀ
1
89

90
DANILOV et al.
serve as an acceptor substrate of a galactosyl phosphate
residue in biochemical reactions of initiation of Oꢀ
antigen polysaccharide biosynthesis catalyzed by
galactosyl phosphotransferase from S. anatum and
S. newport membrane preparations was studied.
RESULTS AND DISCUSSION
11ꢀ[(2ꢀPyridyl)amino]undecyl phosphate (VIII
was obtained using two alternative routes (Scheme 1)
from commercially available 11ꢀbromoundecanol (I)
(method 1) and undecꢀ10ꢀenꢀ1ꢀol (II) (method 2).
)
PhCO H, DBU
2
HO
Br
(I)
HO
OBz
(IV)
1. NaBH , BF
4
⋅ OEt
3 2
OR1
2. H O , NaOAc
2
2
(
(
II): R1 = H
III): R1 = Bz
PhCOCl, Py
PCC
PyNH , NaBH CN
2
3
OBz
O
N
N
OR2
(V)
H
(
VI): R2 = H
VII): R2 = H
NaOH
(
Bu NH PO ,
4
2
4
CCl CN
3
N
N
OPO32–
H
(VIII
)
Scheme 1. The synthesis of 11ꢀ[(2ꢀpyridyl)amino]undecyl phosphate (VIII).
In method 1, 11ꢀbromoundecanol (I) was conꢀ compound (VI) was removed to give 85% 11ꢀ[(2ꢀ
verted to 11ꢀ(benzoyloxy)undecanol (IV) by the treatꢀ pyridyl)amino]undecanꢀ1ꢀol (VII).
ment with benzoic acid in the presence of DBU in
acetonitrile for 3.5 h. The target product was isolated
by column chromatography on silica gel in a yield of
65%.
Phosphorylation of compound (VII) with tetrabuꢀ
tylammonium dihydrogen phosphate in the presence
of trichloroacetonitrile followed by extraction of the
phosphorylation product and its isolation by column
We also developed an alternative method of prepꢀ chromatography on DEAE cellulose DEꢀ52 (АсO–
)
aration of benzoate (IV) starting from undecꢀ10ꢀenꢀ resulted in the target 11ꢀ[(2ꢀpyridyl)amino]undecyl
1ꢀol (II). Compound (II) was treated with benzoyl phosphate (VIII) in a yield of 26%. The structures of
chloride in the presence of pyridine to give a functionꢀ all the new synthesized compounds were confirmed by
ally active alkene (III), which was hydroborated with high resolution ESI mass spectrometry, 1Hꢀ and
diborane generated in situ from NaBH4 and BF3
OEt2 in THF. The intermediate alkyl borates were oxiꢀ mass spectrum of the target compound (VIII) conꢀ
dized in situ with hydrogen peroxide in the presence of tained an signal of 345.1930 corresponding to
aqueous solution of NaOAc to give the target 11ꢀ(benꢀ
+ H]+ for 11ꢀ[(2ꢀpyridyl)amino]undecyl phosꢀ
zoyloxy)undecanol (IV) isolated by column chromaꢀ phate, C16H31O4N2P (
⋅
31P NMR spectrometry and IR spectroscopy. The
m/z
[M
1
m/z 345.1930). In the H NMR
spectrum of this compound, characteristic proton
signals of the proper substituted hydrocarbon radical
tography on silica gel in a yield of 87% (method 2).
Compound (IV) was oxidized to 11ꢀ(benzoylꢀ
oxy)undecanal (V) with PCC in methylene chloride.
After it was isolated by flash chromatography on silica
gel in an argon atmosphere it was subjected to reducꢀ
tive amination with 2ꢀaminopyridine in methylene
chloride in the presence of NaBH3CN. The target
11ꢀ[(2ꢀpyridyl)amino]undecyl benzoate (VI) was isoꢀ
lated in a yield of 85% by crystallization from petroꢀ
leum ether. The use of NaBH(OAc)3 as a reducing
agent resulted under similar conditions in 36% of the
(multiplets at
СH2ОР) and the signals of internal methylene
groups at 1.41–1.22 ppm), and those of the pyridinꢀ
ium residue (a multiplet at 7.72 ppm, a doublet at
δ
3.32 ppm (СH2N) and
δ
3.88 ppm
(
δ
6.95 ppm, and a triplet at 6.77 ppm) were observed.
The P NMR spectrum of compound (VIII) conꢀ
31
tained a single singlet at
istic for phosphomonoesters.
11ꢀBromoundecanol ( ) was also used for the prepꢀ
δ
1.3 ppm, which is characterꢀ
I
target product only. A protective benzoyl group of aration of 11ꢀ[(9ꢀanthracenylcarbonyl)amino]undeꢀ
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THE SYNTHESIS … AND THE STUDY OF ACCEPTOR PROPERTIES
91
cyl phosphate (XVI) (Scheme 2). The treatment of (tetrahydroꢀ
compound ( ) with potassium phtalimide (IX) as dione (XI). It was treated with hydrazine in ethanol in
described in [10] led to 2ꢀ(11ꢀhydroxyundecyl)isoinꢀ an argon atmosphere to give the target 11ꢀ(tetrahydroꢀ
dolinꢀ1,3ꢀdione ( ), the reaction of which with DHP ꢀpyranꢀ2ꢀyloxy)undecylamine (XII) isolated by colꢀ
2Hꢀpyranꢀ2ꢀyloxy)undecyl]isoindolinꢀ1,3ꢀ
I
X
2H
in the presence of РРТS as a catalyst resulted in 2ꢀ[11ꢀ umn chromatography on silica gel in a yield of 90%.
O
(IX)
OR1
Br
OH
N
O
(I)
(X
): R1 = H
XI): R1 = Thp
PPTS, DHP
(
O
N H
2
⋅ H O
4 2
(XIII), DCC
NH2
OThp
N
H
OR2
(
XII)
(
XIV): R2 = Thp
XV): R2 = H
TsOH
(
O
O
Bu NH PO
4
4
2
OPO23–
COOH
N
CCl CN
3
NK
O
H
(
XVI)
(XIII)
(IX)
Scheme 2. The synthesis of 11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl phosphate
(
XVI).
The reaction of the amino derivative (XII) with solubility of compound (XVI) in the salt form, we
9ꢀanthracene carboxylic acid (XIII) in the presence of failed to record satisfactory NMR spectra and use the
DCC in methylene chloride at room temperature in phosphate in biochemical experiments. A more soluꢀ
an argon atmosphere for 15 h resulted in
Nꢀ[11ꢀ(tetꢀ ble dihydro form of compound (XVI) was obtained in
rahydroꢀ ꢀpyranꢀ2ꢀyloxy)undecyl]anthraceneꢀ9ꢀcarꢀ a yield of 41% by acidification to pH ~ 0 of the aqueꢀ
2H
boxamide (XIV) isolated by column chromatography ous suspension of the crystals followed by extraction
in a yield of 50%. A tetrahydropyranyl protective with ethyl acetate. The structure of dihydrophosphate
group was removed in the presence of a catalytic
amount of TsOH in methanol (20 , 3 h).
(
XVI) was confirmed by 1Hꢀ, 13Сꢀ, and 31P NMR and
°C
mass spectrometry. The ESI mass spectrum contained a
signal of 470.2102, which corresponded to
– H]–
470.2102). In
m/z
[M
The obtained 11ꢀ(9ꢀanthracenylcarbonyl)amino]
undecanꢀ1ꢀol (XV) was phosphorylated in 1,2ꢀdichloꢀ
roethane with an excess of tetrabutylammonium dihyꢀ
drogen phosphate and trichloracetonitrile. After the
reaction product was extracted, treated with a mixture
of aqueous NaOH and methanol, and cooled, the preꢀ
cipitate was filtered off and dried to give disodium 11ꢀ
[(9ꢀanthracenylcarbonyl)amino]undecyl phosphate
for phosphate (XVI), С26H34NО5P (m/z
the 1H NMR spectrum of this compound characterisꢀ
tic proton signals of the proper substituted hydrocarꢀ
bon radical (multiplets at
δ
3.77 ppm (СH2N) and
δ
3.43 ppm (СH2ОР) and the signals of internal methylꢀ
ene groups at 1.60–1.20 ppm), and those of the aroꢀ
matic radical at
δ
31P NMR spectrum of compound (XVI) contained a
8.29–7.24 ppm were observed. The
(
(
XVI) as colorless crystals. The structure of compound
XVI) was confirmed by IR and UV spectroscopy and
single singlet at δ 0.9 ppm, which is characteristic for
phosphomonoesters in the H+ꢀform.
mass spectrometry. In the UV spectrum of compound
XVI) the absorption maxima at λmax 250 and 360 nm
The structures of the synthesized nonphosphoryꢀ
lated derivatives (IX)–(XV) were confirmed by IR
spectroscopy, NMR and high resolution ESI mass
spectrometry.
(
were observed, which supported the presence of an
anthracenyl group. The high resolution ESI mass
spectrum contained an
corresponded to
+ H]+ for phosphate (XVI),
С26H32NО5РNa2 (m/z
516.1886). Because of the low as acceptors of the UDPꢀ[14C]Gal galactosyl phosꢀ
m/z signal of 516.1885, which
[M
The ability of phosphates (VIII) and (XVI) to serve
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92
DANILOV et al.
phate residue in the presence of galactosyl phosphoꢀ phate (XVI) and demonstrated their ability to serve as
transferases from S. anatum and S. nеwport cell memꢀ acceptor substrates of the galactosyl phosphate residue
branes was assessed in the reaction with UDPꢀ in the enzymatic reaction catalyzed by galactosyl
[14C]Gal similarly to the protocol for 11ꢀ[(9ꢀanthraꢀ phosphotransferase from Salmonella anatum or Salꢀ
cenyl)methoxy]undecyl phosphate (Scheme 3) [6].
monella newport membrane preparations.
UDPꢀ[14C]Gal
VIII), (XVI
1ꢀPPꢀ(CH2)11ꢀX + UMP
XVII), (XVIII
+
P
ꢀ(CH2)11ꢀХ
→
EXPERIMENTAL
(
)
NMR spectra (
δ
, ppm, J, Hz) of compounds
→
[14C]Gal
(
α
(
VIII) and (XV) were recorded on a Bruker AVANCE
)
IIꢀ600 spectrometer (the working frequency of 600.13
for 1H and 242.93 MHz for 31P); of compound (XVI),
Bruker AMꢀ300 (the working frequency of 75.47 for
(VIII), (XVII) X = PyNH
31
13С and 121.49 MHz for P); and of the other comꢀ
(XVI), (XVIII) X = AntCONH,
Ant = anthracenꢀ9ꢀyl
pounds, Bruker ACꢀ200 (the working frequency of
200.13 for 1H and 121.50 MHz for P). Chemical
31
Scheme 3
.
The enzymatic synthesis of
shifts were measured relative to Mе4Si. High resoluꢀ
tion ESI mass spectra were registered on a Bruker
micrOTOF II spectrometer, at the capillary potential
of 4500 V with a direct (syringe) injection of the samꢀ
1
P
ꢀ[11ꢀ[(2ꢀpyridyl)amino]undecyl]ꢀP2
ꢀ
14
(
α
ꢀDꢀ[ C]galactopyranosyl) diphosphate (XVII) and
1
P
ꢀ{11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl}ꢀ
2
14
P
ꢀ(αꢀDꢀ[ C]galactopyranosyl) diphosphate (XVIII).
ple solution in methanol (3 L/min). The UV specꢀ
μ
trum (λmax, nm) for compound (XVI) was recorded on
The acceptor substrate (VIII) was added to the incuꢀ
bation mixture containing a membrane preparation from
S. anatum cells and radiolabelled UDPꢀ[14C]Gal. After
the incubation, protein coagulation, and coagulate
precipitation by centrifugation the supernatant was
fractioned on a C18 cartridge (DiaPak). The water solꢀ
uble components including the unreacted nucleotide
sugar were eluted with water, and the lipidꢀcontaining
components including the expected product of the
enzymatic reaction, with methanol. The eluates were
analyzed by the TLC technique. We observed on the
plates a radioactive zone with a chromatographic
mobility of Rf 0.80 (D), which corresponded to the
presence of the radioactive galactosyl residue. Also,
upon UV irradiation at a wavelength of 254 nm this
zone fluoresced, which implied the presence of a 2ꢀ
pyridylamino group.
a LKB Biochrom Ultrospec 4050 spectrophotometer;
IR spectrum (
photometer. The solutions were evaporated in vacuum
below 40 . Anionꢀexchange chromatography was
performed on a DEAEꢀcellulose DEꢀ52 (OAc–,
Whatman, England) column (1.2 12 cm). Analytical
ν
, cm–1), on a Specord Mꢀ80 spectroꢀ
°С
×
chromatography was carried out on Silica Gel 60
plates (Merck, Germany) in petroleum ether–
MeOtBu, 1 : 1 (system A), petroleum ether–MeOtBu,
1 : 3 (system B), CHCl3–CH3OH–H2O, 60 : 25 : 4
(system C), and CHCl3–CH3OH–H2O, 10 : 10 : 3
(system D).
The products of enzymatic reactions were analyzed
on DCꢀAlufolien Kieselgel F254 plates (Fluka) in sysꢀ
tems C (for compound (XVI)) or D (for compound
(
XVIII)). The compounds were developed by UV irraꢀ
diation (254 or 360 nm, a Vilber Lourmat lamp,
France), I2 vapor treatment, or 10% H2SO4 in methaꢀ
nol followed by heating. Column chromatography was
In the case of 11ꢀ[(9ꢀanthracenylcarbonyl)amino]
undecyl phosphate (XVI) the incubation mixture conꢀ
tained the S. newport membrane preparation, and the
carried out on a silica gel (40–60
μ
m, Acros Organics)
Bu (a
zone with the chromatographic mobility of Rf 0.60 (C)
column (1.5 30 cm) in petroleum ether–MeO
×
t
was observed on the plates. It was radioactive and fluꢀ
oresced upon UV irradiation at a wavelength of 254
and 360 nm.
linear gradient 9 : 1 to 1 : 1, 60 mL each) with the TLC
control. Reverse phase chromatography was carried
out on C18 cartridges (DiaPak, BioKhimMak, Rusꢀ
Since the zones of the reaction products differed
from those for each of the starting compounds we can
assume with rather a high probability that the interacꢀ
tion of the starting compounds proceeds with the
involvement of galactosyl phosphotransferase from the
membrane preparations and results in the products
shown in Scheme 3.
sia). 11ꢀBromoundecanol (I), undecꢀ10ꢀenꢀ1ꢀol (II),
and 9ꢀanthracene carboxylic acid (IX) were purchased
from Acros Organics. Membrane preparations were
obtained from S. anatum and S. newport cells as
described in [11]. UDPꢀ[14C]Gal, 285 mCi/mmol,
was from Amersham (England) and UDPꢀGal, from
Sigma (United States).
To summarize, we synthesized some derivatives of
11ꢀBenzoyloxyundecanꢀ1ꢀol (IV). Method A.
undecyl phosphate with new fluorescent labels: DBU (2.85 g, 19 mmol) and 11ꢀbromoundecanol (
11ꢀ[(2ꢀpyridyl)amino]undecyl phosphate (VIII) and (3.97 g, 15.8 mmol) were successively added to a susꢀ
11ꢀ[(9ꢀanthracenylcarbonyl)amino]undecyl phosꢀ pension of benzoic acid (2.33 g, 19 mmol) in acetoniꢀ
I)
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THE SYNTHESIS … AND THE STUDY OF ACCEPTOR PROPERTIES
93
trile (15 mL) under stirring at 20
sphere. The reaction mixture was heated at 50
stirred for 3.5 h. The mixture was diluted with MeO
(30 mL), washed with saturated NaHCO3 (2 12 mL), 1520, 1540, 1576, 1584, 1600, 1616, 1700, 1716, 1748,
saturated NaCl (
15 mL), dried with Na2SO4, and 2928, 2856, 3064, 3584, 3648. 1H NMR (CDCl3): 9.76
the solvent was evaporated in vacuum. The residue was (1 H, br s, HC=O), 8.10–7.97 (2 H, m, arom. H),
chromatographed on a silica gel column and the target 7.61–7.36 (3 H, m, arom. H), 4.32 (2 H, t,
11,10 6.6
fractions were concentrated in vacuum to give comꢀ H11), 2.42 (2 H, m, H2), 1.79–1.72 (2 H, m, H3),
pound (IV) (3.0 g, 10.2 mmol, 65%); f 0.52 (A). IR
°
C
in the argon atmoꢀ aldehyde (
V
) (2.21 g, 7.6 mmol, 89%);
R
f 0.70 (A). IR
°C
and (NaCl): 668, 688, 712, 808, 936, 956, 1028, 1072,
Bu 1112, 1176, 1272, 1316, 1388, 1412, 1452, 1464, 1492,
t
×
2
×
J
,
R
1.68–1.60 (2 H, m, H10), 1.48–1.16 (16 H, m, CH2).
ESI mass spectrum: found: 308.2229. Calc. for
C18H26O3, [
+ NH4]+: 308.2220
(NaCl): 668, 688, 712, 1028, 1072, 1112, 1176, 1276,
1316, 1388, 1452, 1464, 1492, 1520, 1584, 1600, 1616,
1700, 1720, 2856, 2924, 3064, 3400, 3584. H NMR
m/z
M
.
1
11ꢀ[(2ꢀPyridyl)amino]undecyl benzoate (VI).
2ꢀAminopyridine (1.89 g, 20 mmol), CH3CООH
(0.6 g, 9 mmol), and NaBH3CN (1.03 g, 16.4 mmol) were
added to a solution of 11ꢀ(benzoyloxy)undecanal (V)
(2.89 g, 10 mmol) in absolute CH2Cl2 (34 mL) and the
(СDCl3): 8.08–8.02 (2 H, m, arom. H), 7.60–7.39
(3 H, m, arom. H), 4.32 (2 H, t,
(2 H, t, 1,2 6.5, H1), 1.85–1.69 (2 H, m, H10), 1.59–
1.52 (2 H, m, H2), 1.43–1.30 (14 H, m, СH2). ESI
mass spectrum: found: 293.2110. Calc. for
C18H28O3 [
+ H]+: 293.2111
J11,10 6.6, H11), 3.63
J
m/z
reaction mixture was stirred for 3.5 h. The mixture was
M
.
washed with 18% HCl (
fractions were neutralized with 6M NaOH to pH 7.0.
The target product was extracted with MeO Bu
(50 mL), washed with water (2 15 mL), saturated
NaCl ( 15 mL), dried with Na2SO4, and the solvent
was evaporated in vacuum. The residue was crystalꢀ
lized from petroleum ether to give compound (VI
(3.16 g, 8.6 mmol, 86%); f 0.31 (B). IR (KBr): 668,
2
×
7 mL). The united aqueous
Method B. A solution of BF3
· Et2O (3.0 g,
22 mmol) in anhydrous THF (15 mL) was added
dropwise under stirring to a suspension of NaBH4
(0.63 g, 16.6 mmol) in anhydrous THF (30 mL) in an
argon atmosphere at 20°C for 15 min. The reaction
mixture was kept for 10 min and undecꢀ10ꢀeneꢀ1ꢀ
benzoate (III) (6.48 g, 23.6 mmol) was added. The
mixture was stirred for 1.5 h, cooled to –10°C and
MeOH (10 mL), 3M NaOAc (10 mL), and 30% H2O2
(4.5 mL) were added dropwise. The mixture was kept
t
×
2
×
)
R
684, 708, 724, 736, 768, 804, 852, 956, 980, 988, 992,
1024, 1068, 1084, 1096, 1108, 1124, 1132, 1160, 1176,
1280, 1292, 1312, 1336, 1368, 1388, 1436, 1452, 1472,
1532, 1572, 1600, 1652, 1716, 1900, 2852, 2924, 2964,
3032, 3088, 3272. 1H NMR (CDCl3): 8.10–8.03 (3 H,
m, arom. H), 7.60– 7.37 (4 H, m, arom. H), 6.55 (1 H,
for 18 h, diluted with MeO
organic layer was washed with water (
urated NaCl ( 25 mL), dried with Na2SO4. The solꢀ
t
Bu (50 mL), and the
2
×
25 mL), satꢀ
2
×
vent was evaporated in vacuum, and the residue was
chromatographed on a silica gel column. The target
fractions were concentrated in vacuum to give comꢀ
pound (IV) (6.21 g, 21.2 mmol, 90%).
m, arom. H), 6.37 (1 H, br d,
J 8.4, arom. H), 4.52
(1 H, br s, NH), 4.32 (2 H, t, 1,2 6.6, H1), 3.29–3.19
J
(2 H, m, H11), 1.84–1.70 (2 H, m, H2), 1.65–1.55
(2 H, m, H10) 1.48–1.30 (14 H, m, CH2). ESI mass
spectrum: found:
m/z 369.2550. Calc. for C23H33N2O2,
.
Undecꢀ10ꢀenyl benzoate (III) [12]. Benzoyl chloꢀ
ride (7.0 g, 50 mmol) and pyridine (3.9 g, 50 mmol)
[M
+ H]+: 369.2537
were added to a solution of undecꢀ10ꢀenꢀ1ꢀol (II
(7.76 g, 45.6 mmol) in anhydrous CH2Cl2 (50 mL).
The reaction mixture was kept for 4 h at 20 , diluted
with MeO Bu (50 mL), washed with water (50 mL),
saturated NaCl ( 25 mL), and dried with Na2SO4
)
11ꢀ[(2ꢀPyridyl)amino]undecanꢀ1ꢀol (VII). Sodium
hydroxide (0.25 g, 6.2 mmol) was added to a solution
of benzoate (VI) (0.76 g, 2.08 mmol) in MeOH
°C
t
(35 mL) and the mixture was stirred for 2 h at 40
Methanol was evaporated in vacuum, and the residue was
dissolved in water (20 mL), extracted with MeO Bu
°C.
2
×
.
The solvents were evaporated in vacuum, and the resiꢀ
t
due was distilled in vacuum to give benzoate (III) (12.0 g,
(50 mL), and dried with Na2SO4. The solvent was
evaporated, and the residue was crystallized from an
ether–petroleum ether mixture to give compound
VII) (0.47 g, 1.8 mmol, 86%); Rf 0.73 (B). IR
(CHCl3): 620, 660, 684, 808, 928, 984, 1048, 1152,
1
43.8 mmol, 97%); bp 125–130
°C
(0.1 mm Hg). H
NMR (СDCl3): 8.09–8.00 (2 H, m, arom. H), 7.62–
7.37 (3 H, m, arom. H), 5.93–5.73 (1 H, m, H10),
5.08–4.88 (2 H, m, H11), 4.33 (2 H, t, J1,2 6.6, H1),
2.11–2.00 (2 H, m, H9), 1.85–1.73 (2 H, m, H2),
1.55–1.33 (14 H, m, СH2).
(
1232, 1288, 1328, 1448, 1476, 1508, 1568, 1600, 1652,
1684, 1700, 1716, 2652, 2856, 2932, 3008, 3032, 3688,
1
11ꢀ(Benzoyloxy)undecanal (V). A suspension of 3136, 3168, 3212, 3304, 3428, 3620. H NMR
PCC (2.76 g, 12.84 mmol) and 11ꢀbenzoyloxyundeꢀ (CDCl3): 8.06 (1 H, d,
canꢀ1ꢀol (IV) (2.5 g, 8.56 mmol) in CH2Cl2 (18 mL) arom. H), 6.55 (1 H, m, arom. H), 6.37 (1 H, d,
was stirred at 20 in an argon atmosphere for 2 h. arom. H), 4.51 (1 H, br t, NH), 3.65 (2 H, m, H11),
J
5.0, arom. H), 7.42 (1 H, m,
J
8.5
,
°C
The precipitate was filtered off through a silica gel 3.29–3.19 (2 H, m, H1), 1.69–1.51 (4 H, m, H2 and
layer and the solvent was evaporated in vacuum to give H10) 1.43–1.30 (m, 14H, CH2). ESI mass spectrum:
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014

94
DANILOV et al.
found:
m
/
z
265.2278. Calc. for C16H29ON2, vacuum to give compound (XI) (1.92 g, 4.78 mmol,
[
M
+ H]+: 265.2274
.
98%); Rf 0.69 (A). IR (NaCl): 668, 720, 816, 868, 904,
988, 1032, 1076, 1120, 1200, 1368, 1396, 1436, 1468,
1540, 1616, 1716, 1772, 2856, 2928. H NMR
(CDCl3): 7.87–7.76 (2 H, m, arom. H), 7.74–7.63
11ꢀ[(2ꢀPyridyl)amino]undecyl phosphate (VIII).
A
1
solution of Bu4NH2PO4 (0.34 g, 1 mmol) in acetoniꢀ
trile (1 mL) and CCl3CN (0.16 g, 1.1 mmol) were
added to a solution of compound (VII) (0.132 g,
0.5 mmol) in anhydrous CH2Cl2 (5 mL) and the mixꢀ
ture was kept for 48 at 20°C and concentrated in vacꢀ
uum. The residue was dissolved in the upper phase of
(2 H, m, arom. H), 4.55 (1 H, t,
3.28 (8 H, m, H11 and H1), 1.88–1.18 (22 H, m,
CH2). ESI mass spectrum: found: 440.2201. Calc.
for C24H35NО4, [
+ K]+: 440.2198
J 3.4, OCHO), 3.91–
m/z
M
.
11ꢀ(Tetrahydroꢀ2 ꢀpyranꢀ2ꢀyloxy)undecylamine
H
the steady BuOH–H2O mixture (7 mL) and washed
(XII). Hydrazine hydrate (1.11 g, 22.2 mmol) was
added to a stirred solution of compound (XI) (0.89 g,
2.22 mmol) in EtOH (45 mL) at 53°C in an argon
with the lower phase of this mixture (
3
×
3 mL). The
upper phase was concentrated in vacuum and the resꢀ
idue was dissolved in MeOH (50 mL). Saturated
ammonia (0.32 mL) was added and the mixture was
atmosphere. The reaction mixture was stirred for 3 h,
filtered, and the filtrate was evaporated in vacuum.
The residue was chromatographed on a silica gel colꢀ
umn. The target fractions were concentrated in vacꢀ
uum to give amine (XII) (0.54 g, 2 mmol, 90%).
IR (NaCl): 668, 724, 816, 868, 904, 988, 1032, 1076,
1120, 1184, 1320, 1352, 1440, 1464, 1648, 1684, 2852,
stirred with Dowex 50
×
8 (NH+4
) (4 g) for 48 h. The
resin was filtered off and the solvent was evaporated in
vacuum. The residue was dissolved in MeOH (12 mL)
and separated into four portions. Each portion was
diluted to 50 mL with MeOH and loaded on a DEAE
cellulose column. The column was washed with methꢀ
anol (30 mL) and eluted with 50 mM CH3COOH in
MeOH (150 mL). The target fractions were united and
evaporated in vacuum to give compound (VIII) (0.28 g,
5924. 1H NMR (CDCl3): 4.55 (1 H, t,
J 3.3, OCHO),
3.87–3.23 (4 H, m, H11 and H1), 2.59 (2 H, t, JNH,1
6.8, H2N), 1.82–1.12 (22 H, m, CH2). ESI mass specꢀ
trum: found:
m/z 272.2595. Calc. for C16H33NО2, [M
80
CD3COOD): 7.72 (2 H, m, arom. H), 6.95 (1 H, d,
9.0, arom. H), 6.77 (1 H, t, 6.6, arom. H), 3.88
μ
mol, 16%); R
f 0.88 (D). 1H NMR (10 : 1 CDCl3)–
+ H]+ 272.2584
.
Nꢀ[11ꢀ(Tetrahydroꢀ2Hꢀpyranꢀ2ꢀyloxy)undecyl]anꢀ
J
J
thraceneꢀ9ꢀcarboxamide (XIV). 9ꢀAnthracene carꢀ
boxylic acid (XIII) (0.36 g, 1.6 mmol) and DCC (0.31 g,
(2 H, m, H1), 3.32 (2 H, m, H11), 1.65 (2 H, m, H2),
1.57 (2 H, m, H10), 1.41–1.22 (14 H, m, CH2).
31P NMR (CDCl3–CD3COOD, 10 : 1): 1.3. ESI mass
1.5 mmol) were added to a stirred solution of amine (XII
)
(0.27 g, 1 mmol) in anhydrous CH2Cl2 (20 mL) at
20°C in an argon atmosphere and the mixture was
stirred for 15 h. The solvents were evaporated, and the
residue was chromatographed on a silica gel column.
The target fractions were concentrated in vacuum to
give amide (XIV) (0.235 g, 0.5 mmol, 50%); mp 80–
spectrum: found:
m
/
z
345.1930
.
.
Calc. for
C16H31O4N2P, [
M
+ H]+: 345.1938
2ꢀ(11ꢀHydroxyundecyl)isoindolinꢀ1,3ꢀdione (X)
[10]. Potassium phtalimide (IX) (1.72 g, 9.3 mmol)
was added to a solution of 11ꢀbromoundecanol (
(2.12 g, 8.45 mmol) in DMF (20 mL) under stirring at
20 . The mixture was kept at 80 for 4.5 h, cooled
to 20 and the precipitate was filtered off. The filtrate
was evaporated to 3 mL, diluted with CH2Cl2 (30 mL),
and the organic layer was washed with water ( 15 mL),
saturated NaCl ( 15 mL), dried with Na2SO4, and
evaporated to give compound ( ) (1.62 g, 5.1 mmol,
60%); mp 84–85 (cf. [10] mp 85–86 C), f 0.33
I)
83°C, Rf 0.47 0.47 (A). IR (KBr): 430, 453, 559, 602,
646, 733, 792, 815, 847, 870, 903, 967, 1023, 1077,
1137, 1155, 1166, 1184, 1201, 1265, 1293, 1355, 1441,
1466, 1553, 1629, 2343, 2361, 2852, 2928, 3055, 3084,
°
C
°
°C
C
1
3262. H NMR (CDCl3): 8.49 (1 H, s, arom. H),
2
×
8.14–7.97 (4 H, m, arom. H), 7.62–7.43 (4 H, m,
2
×
arom. H), 6.08 (1 H, t,
t, 3.4, OCHO), 3.95–3.31 (8 H, m, H1 and H11),
1.91–1.25 (22 H, m, CH2). ESI mass spectrum:
found: 498.2975. Calc. for C31H41О3N, [
Na]+ 498.2979
JNH,1 5.3, OCNH,), 4.58 (1 H,
X
J
°C
°
R
(A). 1H NMR (CDCl3): 7.90–7.78 (2 H, m, arom. H),
7.77–7.65 (2 H, m, arom. H), 3.73–3.59 (4 H, m, H1
and H11), 1.78–1.47 (4 H, m, H2 and H10), 1.45–
1.20 (14 H, m, CH2).
m/z
M +
.
11ꢀ(9ꢀAnthracenylcarbonyl)amino]undecanꢀ1ꢀol (XV).
2ꢀ[11ꢀ(Tetrahydroꢀ2
dolinꢀ1,3ꢀdione (XI). Alcohol (
and dihydropyrane (0.62 g, 7.32 mmol) were added to (0.47 g, 1 mmol) in MeOH (30 mL) at 20
a stirred solution of PPTS (0.125 g, 0.5 mmol) at 20 mixture was stirred for 3 h. The solvent was evapoꢀ
in CH2Cl2 (20 mL). After 15 h, the mixture was conꢀ rated, and the residue was extracted with benzene
centrated to 5 mL, diluted with MeO Bu (30 mL), and (150 mL). The extract was washed with saturated
the precipitate was filtered off. The filtrate was washed NaHCO3 (2 30 mL), water ( 30 mL), and satuꢀ
with water (2 15 mL), saturated NaCl ( 15 mL), rated NaCl ( 30 mL), dried with Na2SO4, and conꢀ
dried with Na2SO4, and the solvent was evaporated in centrated in vacuum. The residue was crystallized from
H
ꢀpyranꢀ2ꢀyloxy)undecyl]isoinꢀ
p
ꢀToluenesulfonic acid TsOH · H2O (0.05 g, 0.26 mmol)
X) (1.55 g, 4.88 mmol) was added to a stirred suspension of amide (XIV
)
°C
and the
°C
t
×
2
×
×
2
×
2
×
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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2014

THE SYNTHESIS … AND THE STUDY OF ACCEPTOR PROPERTIES
95
ethanol to give compound (XV) (0.21 g, 0.53 mmol, (20
53%); mp 142–145 C, Rf 0.67 (MeO
430, 470, 557, 601, 646, 730, 788, 840, 878, 972, 1032, preparation (20
1052, 1073, 1135, 1161, 1255, 1265, 1295, 1428, 1464, ture was incubated for 20 min at 30°C, and nonradioꢀ
μ
L), 0.2 M MgCl2 (0.4
μ
L), UDPꢀ[14C]Gal
°
t
Bu). IR (KBr): (2 nmol, 200000 cpm), and S. anatum membrane
μ
L, 200 g of the protein). The mixꢀ
μ
1544, 1631, 1719, 1788, 2847, 2924, 2936, 3028, 3049, active UDPꢀGal (10 nmol, 2 L) was added for an
μ
3258, 3433. 1H NMR (CDCl3): 8.49 (1 H, s, arom. H), increase in the total product yield. The mixture was
8.10–8.01 (4 H, m, arom. H), 7.54–7.48 (4 H, m, incubated for another 20 min at 25°C, stopped by
arom. H), 6.03 (1 H, br. t, J NH,1 5.3, OCNH), 3.71 (2 cooling to 0°C, diluted with distilled water (0.8 mL),
H, m, H11), 3.64 (2 H, t, J1,2 7.8, H1), 1.75 (2 H, m, and kept for 12 h at 4°C for the protein coagulation.
H2), 1.60–1.30 (16 H, m, CH2). ESI mass spectrum: The protein was precipitated by centrifugation at
found:
392.2584
m
/
.
z
392.2587. Calc. for C26H33NО2, [
M
+ H]+ 10000 rpm and the supernatant was loaded on a C18
cartridge (DiaPak). The cartridge was washed with
water until the radioactivity was eluted (6 mL). The
11ꢀ[(9ꢀAnthracenylcarbonyl)amino]undecyl phosꢀ
phate (XVI). Tetrabutylammonium dihydrogen phosꢀ
phate (0.24 g, 0.7 mmol) and CCl3CN (0.116 g,
0.8 mmol) were added to a stirred solution of alcohol
reaction product was eluted with methanol (5 mL) and
1 mL fractions were collected. The radioactive fracꢀ
tions were united, concentrated in the inert atmoꢀ
sphere to a volume of 100
DCꢀAlufolien Kieselgel F254 plates in system D. Upon
UV irradiation (λ254) the zone with f 0.80 was visualꢀ
ized. The plate was cut in squares of 0.5 0.5 cm,
μL, and analyzed by TLC on
(XV) (0.07 g, 0.178 mmol) in 1,2ꢀdichloroethane
(7 mL) at 50 in an argon atmosphere. In 3 h the
°
C
R
reaction mixture was concentrated in vacuum and the
residue was dissolved in the upper phase of the equilibꢀ
rium BuOH–H2O mixture (4 mL) and washed with
×
immersed into the dioxane ZhSꢀ7 scintillator (Rusꢀ
sia), and the radioactivity was measured on a Deltaꢀ
300 scintillation counter (Tracor Analytic). The analꢀ
ysis showed that the radioactive zone coincided in the
mobility with the fluorescent zone.
the lower phase of this mixture (
4
×
1.5 mL). The
organic phase was evaporated in vacuum, and the resꢀ
idue was dissolved in MeOH (3 mL). Aqueous solution
of NaOH (5 M, 0.08 mL) was added, and the mixture
Acceptor properties of 11ꢀ[(9ꢀanthracenylcarboꢀ
nyl)amino]undecyl phosphate (XVI). The incubation
mixture for the evaluation of acceptor properties of
was kept for 12 h at –18
tered off and dried to give the disodium salt of phosꢀ
phate (XVI) (41 mg); mp 215–217 C, f 0.35 (C). IR
(KBr): 555, 733, 913, 1135, 1234, 1444, 1466, 1525,
1637, 2853, 2926, 3056, 3306, 3406. UV (MeOH),
°C. The precipitate was filꢀ
°
R
compound (XVI) (a final volume of 205
μL) contained
the synthetic acceptor (XVI) (60 nmol) in methanol
(30
(15
μ
L), 50 mM TrisꢀHCl, pH 8.0 (60
μ
L), 0.2 M MgCl2
λmax
found:
H]+ 516.1886
(
ε
): 250 (65500), 360 (4625). ESI mass spectrum:
μ
L), UDPꢀ[14C]Gal (30 nmol, 100000 impulse/min,
m/z
516.1885. Calc. for C26H32NNa2О5Р, [ M +
Amersham, England), and S. newport membrane
.
preparation (70 L, 200 g of the protein). The mixꢀ
μ
μ
The resulting crystals were suspended in water
(7 mL), acidified with concentrated HCl to pH ~ 0
and extracted with ethyl acetate (30 mL). The extract
was washed with saturated NaHCO3 (2 mL), water
(2 7 mL), and saturated NaCl ( mL), dried with
ture was incubated for another 30 min at 33°C, and
treated as described above. The methanol fractions
bearing radioactivity were united, concentrated in the
inert atmosphere, and analyzed by TLC in system D.
Upon UV irradiation (λ254 or λ360) the zone with Rf
0.60 was visualized. The analysis of the chromatoꢀ
graphic plates showed that the radioactive zone coinꢀ
cided in mobility with the fluorescent zone.
,
×
7
×
2 7
×
Na2SO4, and concentrated in vacuum to give dihydroꢀ
phosphate (XVI) (34 mg, 0.073 mmol, 41%); Rf 0.33
(C). 1H NMR (CDCl3–CD3OD, 2 : 1): 8.29 (1 H, s,
arom. H), 7.90–7.75 (4 H, m, arom. H), 7.38–7.24
(4 H, m, arom. H), 3.77 (2 H, m, J11, 10 6.63, H11),
3.43 (2 H, m, J1,2 7.1, H1),1.60–1.38 (4 H, m, H2 and
ACKNOWLEDGMENTS
H10), 1.27 (14 H, m, CH2). 13C NMR (2 : 1 CDCl3–
CD3OD): 128.21, 127.83, 126.36, 125.21, 124.58,
66.58, 39.93, 30.08, 29.99, 29.26, 29.19, 29.11, 28.98,
28.90, 26.81, 25.18. 31P NMR (2 : 1 CDCl3–CD3OD):
The work was supported by the Russian Foundaꢀ
tion for Basic Research, project no.13ꢀ04ꢀ00358.
0.9. ESI mass spectrum: found:
m/z 470.2102. Calc.
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μ
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014

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Translated by E. Shirokova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014