11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d- glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59

Article abstract of DOI:10.1016/j.carres.2010.09.021

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P¹-11-phenoxyundec

Full text of DOI:10.1016/j.carres.2010.09.021

Carbohydrate Research 345 (2010) 2636–2640
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-a-D-glucopyranosyl
phosphate acceptor in O-antigen repeating unit assembly of Salmonella
arizonae O:59 q
⇑
Tatyana N. Druzhinina , Leonid L. Danilov, Vladimir I. Torgov, Natalya S. Utkina, Nadezhda M. Balagurova,
Vladimir V. Veselovsky, Alexander O. Chizhov
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2010
Received in revised form 15 September 2010
Accepted 18 September 2010
Available online 25 September 2010
A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transfer-
ase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on
lipid-phosphate) into P1-11-phenoxyundecyl, P2-2-acetamido-2-deoxy-
are described.
a-D-glucopyranosyl diphosphate
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
11-Phenoxyundecyl phosphate
Lipid acceptor
Glycosylphosphate transferase
O-Antigen biosynthesis
Salmonella arizonae
1. Introduction
the acceptor properties of 11-phenoxyundecyl phosphate 3 itself
remained unknown.
Most bacteria produce and utilise undecaprenyl phosphate and
its glycosyl esters as substrate acceptors for glycosyltransferases
involved in the assembly of glycopolymers such as peptidoglycans
and lipopolysaccharides.
Due to this fact, we decided to investigate acceptor properties of
phosphate 3 in biosynthesis of 6 catalysed by the corresponding
enzyme from Salmonella arizonae O:59.
The required 11-phenoxyundecyl dihydrogen phosphate has re-
cently been synthesised4 by a multistep procedure.
In the present work, an alternative method for the synthesis of
phosphate 3 is reported.
The activity of 11-phenoxyundecyl phosphate 3 as a substrate
acceptor was tested with crude GlcNAc-P transferase from S. arizo-
nae and the structure of the reaction product prepared by biosyn-
thesis was proved by comparison with a synthetic sample of 6b.
Low content of polyprenyl phosphate in bacterial cells and high
lability make its isolation an extremely labour-consuming proce-
dure and significantly hamper its application in biochemical studies.
It was found2,3 that bacterial polyprenols can efficiently be
substituted in biochemical reactions by structurally close plant ana-
logues, both native and synthetic. The accessibility of plant polypre-
nols and the development of efficient methods of their chemical
phosphorylation2,3 gave an impetus to systematic investigations of
the structure–function relationships within polyprenyl phosphates.
Recently, a new type of lipid phosphate (compound 3) and lipid
diphosphate sugar 6 have been synthesised, which contained an
11-phenoxyundecyl group instead of the polyprenyl chain.4
Diphosphate 6 was shown to be able to replace a polyprenyl-con-
taining substrate in an assay for b-1,3-galactosyltransferase activ-
ity from a series of Escherichia coli VW187 strains.4,5 However,
2. Results and discussion
Synthesis of the target compound 3 is presented in Scheme 1.
11-Phenoxyundecanoic acid 1 was prepared as described.6 Acid
1 was smoothly reduced with diborane generated in situ to give
11-phenoxyundecan-1-ol 2, which was phosphorylated with POCl3
in the presence of triethylamine in dry THF affording 11-phenoxy-
undecyl dihydrogen phosphate 3. As judged by 1H NMR spectra,
compounds 2 and 3 were identical to the corresponding com-
pounds described earlier.4
q
Preliminary communication see Ref. 1.
The transformation of phosphate 3 catalysed by transferase from
S. arizonae was anticipated to afford diphosphate 6. We undertook
⇑
Corresponding author. Tel.: +7 499 1377570; fax: +7 499 1355328.
E-mail address: druzh@ioc.ac.ru (T.N. Druzhinina).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.09.021

T. N. Druzhinina et al. / Carbohydrate Research 345 (2010) 2636–2640
2637
b
a
CO2H
PhO
OH
PhO
PhO
2
1
OPO3H2
3
Scheme 1. Reagents and conditions: (a) BF3ꢁEt2O/NaBH4/THF (anhyd), 2: yield 83%; (b) (i) POCl3, Et3N/THF (anhyd), (ii) NaOH (aq), (iii) HCl (aq), 3: yield 70%.
attempts to synthesise this compound as a reference substance by a
procedure different from that described.4 The idea of our approach7
of 6 reported earlier.4 The structure of 6b was confirmed by
high-resolution ESI mass-spectra (HRESIMS).
was the use of 2-acetamido-2-deoxy-
phate 5 for the coupling with phosphate 3 (Scheme 2).
2-Acetamido-2-deoxy- -glucopyranosyl phosphate 5 was pre-
pared by fusion of 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-
-glucopyranose with dioxane diphosphate in vacuo followed by
a
-
D
-glucopyranosyl phos-
It is necessary to underline that sodium salt 6b could be stored
for at least 6 months at +20 °C and is much more stable than pyrid-
inium salt of 6 (cf. Ref. 4).
a-D
a
-
The O-antigen of S. arizonae O:59 has a trisaccharide repeating
D
unit with the following structure:10 ?b-
D-Galp-(1?3)-a-L-Fucp-
deacetylation, it was isolated by anion-exchange chromatography
NAc-(1?3)-b-D-GlcpNAc-(1?.
as described.8
It is known that biochemical synthesis of the O-antigen repeat-
ing unit starts with formation of GlcNAcPP Pre from UDP-GlcNAc
and polyprenyl phosphate 8 (reaction A). So far, the possibility of
substitution of 3 for 8 has not been investigated. Therefore, we as-
sayed 11-phenoxyundecyl phosphate (PhU-P) 3 as a putative accep-
tor in the initiation reaction of the O-antigen repeating unit
assembly catalysed by UDP-GlcNAc:polyprenyl phosphate GlcNAc-
phosphotransferase from S. arizonae O:59 (Scheme 2; reaction B).
Triethylammonium phosphate 3 was treated with an excess of
1,10-carbonyldiimidazole (CDI) giving 11-phenoxyundecyl phos-
phoroimidazolidate 4, which reacted with triethylammonium gly-
cosyl phosphate 5. An attempt to isolate the target compound 6 by
anion-exchange chromatography, as described by us earlier,7,8 was
unsuccessful due to almost no separation of the reaction products.
Previously, we have isolated polyprenyl diphosphate sugars in a
pure state by ion-exchange chromatography. The failure to isolate
11-phenoxyundecyl-containing diphosphate 6 by ion-exchange
chromatography as individual compound is presumably due to
aggregation of the n-alkyl part of the molecule.
Therefore, compound 6 (as ammonium salt 6a) was isolated by
reversed-phase chromatography on C18 Sep-Pak cartridge with a
yield of 25% using a procedure similar to that employed for purifi-
cation of a decyl analogue of 6.9
According to our knowledge sodium salts of diphosphate com-
pounds are much more stable than ammonium salts. Therefore,
we synthesised 6b by a procedure described in Ref. 4 and obtained
6 by deacetylation of compound 74 with sodium methoxide in
methanol followed by neutralisation with AcOH and isolation of
sodium salt 6b by reversed-phase chromatography on C18 Sep-
Pak cartridge. Structures of 6a and 6b were confirmed by 1H and
31P NMR spectra, which were similar to those for pyridinium salt
The reaction of compound 3 with UDP-a-D
-[14C]-GlcNAc is pre-
sented in Scheme 3 (reaction B).
After incubation of 3 with UDP-[14C]GlcNAc and an membrane
preparation containing glycosyl transferases (cytoplasmic mem-
branes prepared according procedure described in Ref. 11) from
S. arizonae O:59, the reaction mixture was deproteinised and ap-
plied to a C18 Sep-Pack cartridge. Unconsumed UDP-[14C]GlcNAc
was eluted with water and lipid-linked products were eluted with
methanol. The eluates were analysed by TLC (Fig. 1).
The UV-absorbing, radioactive zone 1 (Fig. 1) with mobility of
the synthetic sample of 6b was eluted with MeOH and the eluate
was analysed by mass spectrometry. High-resolution ESI mass-
spectra (HRESIMS): m/z 626.2137 (calcd for C25H43NO13P2 [MꢀH]ꢀ
626.2137).
It has previously been demonstrated that moraprenyl phos-
phate 8, the phosphate of C50-C60-polyprenols from mulberry
HOCH2
HO
HO
AcHN
O
P
c
O
N
PhO
O
3
N
4
O-
2-
OPO3
5
d
HOCH2
HO
HO
O
O
O
P
-O
P
O-
AcHN
O
O
O
OPh
6a,b
e
AcOCH2
O
AcO
AcO
O
O
P
-O
P
O-
AcHN
O
O
O
OPh
7
Scheme 2. Reagents and conditions: (c) (i) Et3N, (ii) 1,1-CDI/THF, (iii) MeOH; (d) (i) THF/DMSO, (ii) DE-52 (OAcꢀ)/MeOH–NH4OAc, (iii) C18 Sep-Pak/MeOH-water, ammonium
salt 6a: yield 25%; (e) (i) MeONa/MeOH, (ii) AcOH, (iii) C18 Sep-Pak/MeOH–water, sodium salt 6b: yield 96%.

2638
T. N. Druzhinina et al. / Carbohydrate Research 345 (2010) 2636–2640
O
CH2OH
O
HN
O
O
O
OH
N
O
NHAc
P
O-
O
P
O-
O
HO
-2
O
R
OPO3
+
OH
OH
O
N
CH2OH
O
HN
O
O
P
O
O
OH
-O
O
HO
O
P
O
P
OR
+
O
NHAc
O-
O-
O-
Target product
OH
OH
(Reaction A)
R=
8
3
6-8
(Reaction B)
R=
PhO
3
Scheme 3. Enzymatic synthesis of lipid diphosphate a-D
-[14C]GlcNAc from natural (reaction A) and synthetic (reaction B) acceptors.
(Morus alba) leaves containing C55-polyprenol as the main compo-
nent,3 and plant C55-polyprenyl phosphate 9 can substitute effi-
ciently bacterial undecaprenyl phosphate 10 (Chart 1) in
biochemical assays with a number of Salmonella strains3 and other
bacteria.12–14. The acceptor efficiency of compound 3 was com-
pared with that of compound 8.
testing enzymes involved in the assembly of O-antigen repeating
units. Sodium salt of 6 (Scheme 1) is much more storage-stable
than the pyridinium salt (storage without decomposition con-
trolled by NMR for more than one year at ꢀ20 °C and at least
3 months at +20 °C).
Judging from the radioactivity of the main product (reactions A
and B, Scheme 2), the relative efficiency of phosphate 3 as the
3. Experimental
acceptor of 2-acetamido-2-deoxy-a-D-glucopyranosyl phosphate
3.1. General methods
was estimated as ꢂ30% compared with that of moraprenyl phos-
1H and 31P NMR spectra were recorded on Bruker AC-200 and
Bruker AMX-600 spectrometers. High-resolution ESI mass-spectra
(HRESIMS) were recorded on a micrOTOF II (Bruker Daltonics)
spectrometer at a capillary potential of +3200 V (negative ion
phate 8 (Scheme 2).
The data obtained show that 11-phenoxyundecyl phosphate 3
can substitute moraprenyl derivative (8, Chart 1) as a glycosyl
phosphate acceptor in biochemical reactions and is suitable for
800
600
400
200
0
(1)
0.0
0.1
0.2
0.3
0.4
0.5
Rf
0.6
0.7
0.8
0.9
1.0
Figure 1. Analysis of products of the enzymatic reaction of 3 with UDP-a-D-GlcNAc by TLC in CHCl3–MeOH–H2O, 10:10:3 (system B). The mobility of the main radioactive
product (1) coincides with that of an authentic synthetic sample of 6a (Rf 0.73).

T. N. Druzhinina et al. / Carbohydrate Research 345 (2010) 2636–2640
2639
1H NMR (200,13 MHz, CDCl3–CD3OD, 5:1) d: 1.05–1.75 (m, 18H,
9CH2), 3.79–3.90 (m, 4H, CH2-1, CH2-11), 6.86–6.98 (m, 2H, aro-
matic protons), 7.22–7.35 (m, 2H, aromatic protons); 31P NMR
(242.94 MHz, CD3OD) d: 0.5 (s) (cf. Ref. 4).
2-
OPO3
m
n
8; m=3, n=6-8
9; m=3, n=7
10; m=2, n=8
3.4. Synthesis of P1-11-phenoxyundecyl, P2-2-acetamido-2-
deoxy-a-D-glucopyranosyl diphosphate (6)
Chart 1. Chemical structure of polyprenyl phosphates: 8: moraprenyl phosphate;
9: plant undecaprenyl phosphate; 10: bacterial undecaprenyl phosphate.
3.4.1. Ammonium salt (6a)
To phosphate 3 (26 mg, 0.075 mmol), an excess of triethylamine
(0.5 mL) and dry toluene (3 mL) were added and the mixture was
concentrated to dryness. The residue of triethylammonium salt
was dissolved in MeOH (0.1 mL), dry benzene (3 mL) was added
and the mixture was lyophilised. Then CDI (98 mg, 0.73 mmol)
and dry THF (2 mL) were added under argon and the solution
was stirred at room temperature for 2 h. Analysis by TLC (system
B) showed the conversion of 3 (Rf 0.85, system B) into 11-phenoxy-
undecyl phosphoroimidazolidate 4 (Rf 0.90, system B).
mode) and ꢀ4500 V (positive ion mode) with syringe injection of a
methanolic solution of a sample (3 mL/min). Analytical TLC was
performed on glass plates with Silica Gel 60 G, Silica Gel 60 F254
(E. Merck) or on Silica Gel/TLC cards DC-Alufolien-Kieselgel F254
(Fluka) in CHCl3–CH3OH–H2O, 60:25:4 (system A), 10:10:3 (system
B) or 60:40:5 (system C). Compounds containing PhO-group were
visualised on TLC plates in UV light. Phosphoric esters were de-
tected by spraying the plate with a universal reagent for phospho-
lipids15 followed by heating on a hot plate.
MeOH (0.2 mL) was added to the mixture and stirring was con-
tinued for 1 h. The solution was concentrated in vacuum and the
residue was dissolved in dry THF (0.3 mL) under argon. 2-Acetam-
UDP-
Amersham.
a-D lCi/lmol) was purchased from
-[14C]-GlcNAc (261
ido-2-deoxy-a-D
-glucopyranosyl phosphate 58 (triethylammo-
The TLC cards were cut in pieces of (0.5 ꢃ 1 cm) and the radio-
activity was counted with a dioxane scintillator ZhS-50 (Russian
Federation) on a Delta-300 liquid scintillation counter (Tracor
Analytic). Mini-column reversed-phase chromatographic separa-
tions were performed on C18 Sep-Pak cartridges (Waters). Anion-
exchange chromatography was performed on a column (1.2 ꢃ
12 cm) with DEAE-cellulose DE-52 (OAcꢀ, Whatman). Reagent
grade solvents were dried and distilled before use according to
standard procedures.
nium salt, 40 mg, 0.094 mmol) was dissolved in MeOH (0.1 mL),
mixed with dry benzene (3 mL) and lyophilised. The residue was
dissolved in dry DMSO (0.4 mL) and the solution was added to
the activated lipid phosphate 4. The reaction mixture was stirred
under argon for 48 h at room temperature, diluted with MeOH
(25 mL) and applied onto a column with DEAE-cellulose DE-52
(OAcꢀ). The column was eluted with MeOH (25 mL), 0.05 M
NH4OAc in MeOH (100 mL), 0.08 M NH4OAc in MeOH (100 mL)
and 0.2 M NH4OAc in MeOH (50 mL) and fractions (6 mL) were col-
lected. Fractions containing compound 6 (TLC: on glass plates Rf
0.10, system A; 0.65, system B; on aluminium cards Rf 0,74, system
B) (with admixture of nonseparated by-products) were pooled,
concentrated to dryness, MeOH was repeatedly added to, and dis-
tilled from, the residue (10 mL ꢃ 5) and the residue was dissolved
in water (15 mL). Aliquots of the solution (5 mL) were applied on
preactivated C18 Sep-Pak cartridge and elution was carried out
with water (10 mL), 5% MeOH in water (10 mL) and 10% MeOH
in water (10 mL). The latter eluate contained 6 (TLC). Portions of
the eluate containing chromatographically homogeneous 6 were
collected and concentrated in vacuum to dryness. The residue
was dissolved in dry benzene (5 mL) and lyophilised affording
ammonium salt 6a as a white amorphous solid (11 mg, 25%); Rf
0.10 (system A), 0.65 (system B) on glass plates or 0.74 (system
B) on aluminium cards. 1H NMR (600.13 MHz, CD3OD) d: 1.30–
1.45 (m, 12H, CH2), 1.51 (m, 2H, CH2), 1.68 (m, 2H, CH2), 1.78 (m,
2H, CH2), 2.07 (s, 3H, CH3CO), 4.14–3.66 (m, 10 H, H-2, H-3, H-4,
H-5, H-6, H-60, CH2OP, CH2OPh), 5.55 (dd, 1H, J1,2 3.0 Hz, J1,P
7.0 Hz, H-1), 6.90 (m, 3H, aromatic protons), 7.26 (m, 2H, aromatic
protons); 31P NMR (242.94 MHz, CD3OD) d: ꢀ10.1 (br, P-2), ꢀ12.3
(br, P-1).
3.2. Synthesis of 11-phenoxyundecan-1-ol (2)
A solution of BF3ꢁEt2O (1.31 g, 9.2 mmol) in dry THF (5 mL) was
added slowly to a stirred suspension of NaBH4 (0.38 g, 10 mmol) in
dry THF (20 mL) at 10 °C in an atmosphere of argon. After 10 min,
acid 16 (1.66 g, 6 mmol) was added in portions (of about 0.2 g) over
10 min. The resulting mixture was stirred for 4 h and kept over-
night at room temperature, diluted with MeOBut (50 mL), washed
with water (5 ꢃ 10 mL), saturated aqueous NaHCO3 (10 mL), brine
(2 ꢃ 10 mL), dried with anhydrous Na2SO4, and the filtrate was
concentrated to dryness. The residue was crystallised from petro-
leum ether affording alcohol 2 (1.32 g, 83%); white solid; mp 55–
57 °C. 1H NMR (200,13 MHz, CDCl3) d: 1.22–1.88 (m, 18H, 9CH2),
3.65 (t, 2H, J1,2 6.5 Hz, H2C-1), 3.97 (t, 2H, J10,11 6.5 Hz, H2C-11),
6.86–6.98 (m, 3H, aromatic protons), 7.22–7.35 (m, 2H, aromatic
protons) (cf. Ref. 4).
3.3. Synthesis of 11-phenoxyundecyl dihydrogen phosphate (3)
A solution of 11-phenoxyundecan-1-ol (2) (0.27 g, 1.02 mmol)
in dry THF (5 mL) was added dropwise to a stirred solution of POCl3
(0.34 g, 2.2 mmol) and Et3N (0.22 g, 2.2 mmol) in dry THF (5 mL) at
room temperature under argon. The mixture was stirred for 1 h
and added to an intensively agitated mixture of 10% aqueous NaOH
(6 mL) and THF (20 mL). The heterogeneous system was stirred
vigorously for 3 h at room temperature and THF was evaporated.
PriOH (10 mL) was added to the residual aqueous solution and
after agitation the system was concentrated in vacuum. The resi-
due was solubilised in H2O (10 mL) and 10% aqueous HCl (7 mL)
was added. The emulsion obtained was extracted with MeOBut
(30 mL), the organic phase was washed with brine, dried with
anhydrous Na2SO4 and the filtrate was concentrated to dryness.
The residue was crystallised from MeCN affording phosphate 3
(0.24 g, 70%). Rf 0.20 (system A), 0.85 (system B) on glass plates.
3.4.2. Sodium salt (6b)
Compound 7 was synthesised from 11-phenoxyundecyl phos-
phate 3 (obtained as described in Section 3.3) and 2-acetamido-
2-deoxy-3,4,6-tri-O-acetyl-a-D-glucopyranosyl phosphate as de-
scribed previously.4
Methanolic MeONa (2 M) was added to a stirred solution of 7,
ammonium salt (0.025 g, 0.032 mmol), in MeOH (5 mL) to pH 12.
The reaction mixture was agitated until O-deacetylation was com-
plete (control by TLC, system C), neutralised with AcOH and con-
centrated in vacuum. The residue was dissolved in H2O (20 mL)
and passed through a preactivated C18 Sep-Pac cartridge, which
was washed with H2O (100 mL) and MeOH (20 mL). The methano-
lic eluate was concentrated, the residue was dissolved in MeOH

2640
T. N. Druzhinina et al. / Carbohydrate Research 345 (2010) 2636–2640
(0.5 mL), benzene (5 mL) was added and solution was lyophilised
affording sodium salt 6b as a white amorphous solid (0.020 g,
96%); Rf 0.65 (system B), 0.25 (system C) on glass plates or 0.75
(system B) on TLC cards. 1H NMR (600.13 MHz, CD3OD) d: 1.30–
1.45 (m, 12H, 6CH2), 1.50 (m, 2H, CH2), 1.65 (m, 2H, CH2), 1.76
(m, 2H, CH2), 2.05 (s, 3H, CH3CO), 3.35 (t, 1H, J3,4 10.0 Hz, J3,2
ing radioactivity. The reaction product was eluted with MeOH
(1 mL ꢃ 5), methanolic eluate was concentrated and analysed on
TLC cards (system B) with reference to diphosphate 6b. The radio-
active UV-absorbing zone from the TLC plate that contained the
biochemically synthesised
a-D
-[14C]GlcNAc-PP-PhU, which coin-
cided in mobility with that of the authentic sample of 6b, was
eluted with MeOH and HRESIMS of the eluate confirmed the
molecular mass of the reaction product (calcd for C25H43NO13P2
[MꢀH]ꢀ 626.2137; found m/z 626.2137).
10.0 Hz, H-3), 3.65 (dd, 1H, J5,6 6.0 Hz, J6,6 12.0 Hz, H-60), 3.75 (t,
0
0
1H, J4,3 10.0 Hz, J4,5 10.0 Hz, H-4), 3.92 (dd, 1H, J6,6 12.0 Hz, J6,5
0
2.0 Hz, H-6), 3.94 (t, 2H, JH,H 6.5 Hz, CH2OPh), 3.97(m, 2H, CH2OP),
3.98 (m, 2H, H-2, H-5), 5.55 (dd, 1H, J1,2 2.9 Hz, J1,P 6.8 Hz, H-1),
6.88 (m, 3H, aromatic protons), 7.23 (m, 2H, aromatic protons);
13C NMR (150.90 MHz, CD3OD) d: 174.5, 160.7, 130.5, 121.6,
115.7 (aromatic carbons), 96.3 (d, J1,P1 3.8 Hz, C-1), 75.1 (C-5),
77.1 (C-4), 72.5 (C-3), 69.0 (CH2OPh), 67.5 (d, JC,P2 3.3 Hz, CH2OP),
63.1 (C-6). 55.5 (d, J2,P1 7.7 Hz, C-2), 32.0(d, JC,P2 5.8 Hz, CH2CH2OP),
31.9, 30.9, 30.8, 30.7, 30.6, 30.5, 27.3, 27. 1, 23.2, 19.6 (aliphatic
carbons) ; 31P NMR (242.94 MHz, CD3OD) d: ꢀ10.7 (d, JP2,P118.0 Hz,
P-2), ꢀ12.2 (d, JP1,P218.0 Hz, P-1); HRESIMS: calculated for
C25H43NO13P2Na [M+H]+: m/z 650.2102; found: m/z 650.2069.
Acknowledgement
This work was supported by the Russian Foundation for Basic
Research (Project 08-04-00556).
References
1. Danilov, L. L.; Veselovsky, V. V.; Balagurova, N. M.; Druzhinina, T. N. Russ. J.
Bioorg. Chem. 2009, 35, 391–392.
2. Veselovsky, V. V.; Grigorieva, N. Y.; Moiseenkov, A. M. Chem. Phys. Lipids 1989,
51, 147–157.
3. Danilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Maltsev, S. D.; Shibaev, V. N.
Chem. Phys. Lipids 1989, 51, 191–203.
3.5. Biochemical assays
4. Montoya-Peleaz, J. P.; Riley, J. G.; Szarek, W. A.; Valvano, M. A.; Schutzbach, J. S.;
Brockhausen, I. Bioorg. Med. Chem. Lett. 2005, 15, 1205–1211.
5. Riley, J. G.; Menggad, M.; Montoya-Peleaz, P. J.; Szarek, W. A.; Marolda, C. L.;
Valvano, M. A.; Schutzbach, J. S.; Brockhausen, I. Glycobiology 2005, 15, 605–
613.
6. Paulshock, M.; Moser, C. M. J. Am. Chem. Soc. 1950, 72, 5073–5077.
7. Danilov, L. L.; Shibaev, V. N.. In Studies in Natural Products Chemistry; Atta-ur-
Rahman, Ed.; Elsevier: Amsterdam, Oxford, New York, Tokyo, 1991; Vol. 8, pp
63–114.
8. Danilov, L. L.; Maltsev, S. D.; Shibaev, V. N. Bioorg. Khim. 1986, 12, 488–492.
9. Brockhausen, I.; Larrson, E. A.; Hindsgaul, O. Bioorg. Med. Chem. Lett. 2008, 18,
804–807.
10. Vinogradov, E. V.; Knirel, Y. A.; Lipkind, G. M.; Shashkov, A. S.; Kochetkov, N. K.;
Stanislavsky, E. S.; Kholodkova, E. V. Bioorg. Khim. 1987, 13, 1275–1281.
11. Shibaev, V. N.; Kusov, Y. Y.; Druzhinina, T. N.; Kalinchuk, N. A.; Kochetkov, N.
K.; Kilesso, V. A.; Roshnova, S. S. Bioorg. Khim. 1978, 4, 47–56.
12. Rush, J. S.; Rick, P. D.; Weachter, C. J. Glycobiology 1997, 7, 315–322.
13. Al-Dabbagh, B.; Mengin-Lecreulx, D.; Bouhss, A. J. Bacteriol. 2008, 190, 7141–
7146.
The incubation mixtures for testing compound
ammonium salt) contained (in a total volume of 80
of phosphate 3 or moraprenyl phosphate 8 (ammonium salt) (final
concentration 0.5 mM); 30 L of 0.1 M Tris–HCl buffer, pH 8.0 (fi-
nal concentration 35 mM); 4 L of 0.2 M MgCl2 (final concentration
10 mM); 15 L of 0.5 % aqueous Tween 85 and 10 L of MeOH. The
mixture was Vortexed for 60 s and UDP-
-[14C]-GlcNAc was
3
(P-PhU,
l
L) 40 nmol
l
l
l
l
a
-D
added (1.5 nmol, 100,000 cpm for radioactive substrate).
Membrane preparation (prepared according to procedure de-
scribed in Ref. 11) had been added at the last moment (20
ꢂ20 of protein). After incubation of probes at 35 °C for 45 min
with gentle mixing, cold distilled water was added up to the final
volume of 400–500 L and the test tubes were kept at 0–4 °C for
lL,
c
l
12–20 h. Coagulated protein was removed by centrifugation
(8000 rpm, 10 min), the supernatant was applied on a C18 Sep-
Pak cartridge, the excess of nucleotide sugar was eluted with water
(1 mL ꢃ 5), completeness of its removal was monitored by measur-
14. Al-Dabbagh, B.; Blanot, D.; Mengin-Lecreulx, D.; Bouhss, A. Anal. Biochem. 2009,
391, 163–165.
15. Vaskovsky, V. E.; Kostetsky, E. Y.; Vasendin, I. M. J. Chromatogr. 1975, 114, 129–
141.