Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104

Article abstract of DOI:10.1016/j.carres.2017.10.016

Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P¹-(11-phenoxyundecyl)-P²- (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P¹-(11-phenoxyundecyl)-P²-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively.

Full text of DOI:10.1016/j.carres.2017.10.016

Carbohydrate Research 453-454 (2017) 19e25
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-
a
-D-rhamnopyranosyl-
phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-
galactopyranosyl- -D-galactopyranosyl) diphosphate for the
a
-D-glucopyranosyl) diphosphate and P1-(11-
b
-D-
a
investigation of biosynthesis of O-antigenic polysaccharides in
Pseudomonas aeruginosa and Escherichia coli O104
,
Vladimir Torgov a, Leonid Danilov a, Natalia Utkina a *, Vladimir Veselovsky a,
Inka Brockhausen b
a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
b Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada
a r t i c l e i n f o
a b s t r a c t
Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P1-(11-
Article history:
phenoxyundecyl)-P2- (2-acetamido-2-deoxy-3-O-
a
-D-rhamnopyranosyl-
a
-D-glucopyranosyl) diphos-
Received 15 September 2017
Received in revised form
24 October 2017
phate
and
P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-
b
-D-galactopyranosyl- -D-gal-
a
actopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic
polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively.
© 2017 Elsevier Ltd. All rights reserved.
Accepted 24 October 2017
Keywords:
P1- (11-phenoxyundecyl) -P2-(2-acetamido-
2-deoxy-3-O-
glucopyranosyl) diphosphate
P1-(11-phenoxyundecyl) -P2-(2-acetamido-
2-deoxy-3-O- -D-galactopyranosyl- -D-
a-D-rhamnopyranosyl-a-D-
b
a
galactopyranosyl) diphosphate
Chemical synthesis
Enterobacterial glycosyltransferases
Pseudomonas aeruginosa
Escherichia coli O104
1. Introduction
practically impossible due to their extremely low concentrations
and short lifetime in bacterial cells. Data concerning the possibility
The biosynthesis of enterobacterial O-antigenic polysaccharides
occurs via intermediate formation of polyprenyl diphosphate
sugars anchored into the cell membrane. The assembly of the
repeating units of the polysaccharides is catalyzed by specific gly-
cosyltransferases. Elucidating the mechanisms of this process
opens a way to development of a new antibacterial drug generation.
The use of native polyprenyl diphosphate sugars for this purpose is
of replacing polyprenyl group with phenoxyundecyl group in the
abovementioned acceptor substrates have been published reсently
[1].
We have previously synthesized a number of phenoxyundecyl
diphosphate monosaccharides Sug-PP-(CH2)11OPh (Sug ¼ GlcNAc,
GalNAc, Glc or Gal). It was demonstrated that these synthetic an-
alogues can function as acceptor substrates for the corresponding
bacterial glycosyltransferases catalysing the second step of O-
antigenic polysaccharide repeating unit biosynthesis [1e7].
This work is a continuation of our research on the chemical
synthesis and investigation of the biological properties of non-
* Corresponding author.
E-mail address: utkinans@yahoo.com (N. Utkina).
https://doi.org/10.1016/j.carres.2017.10.016
0008-6215/© 2017 Elsevier Ltd. All rights reserved.

20
V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
isoprenoid analogues of undecaprenyl diphosphate sugars. Two
new compounds of this series contain a phenoxyundecyl residue
instead of undecaprenyl, and the disaccharide residue as the sugar
moiety: P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-
a
-D-rhamnopyranosyl-
a
-D-glucopyranosyl)
diphosphate
and
[D-
Rha( 1/3)-D-GlcNAc
a
a
-PP-UnOPh, 1]
P1-(11-
phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-
anosyl-
NAc -PP-UnOPh, 2]. These compounds were synthesized to
b-D-galactopyr-
a
-D-galactopyranosyl) diphosphate [D-Gal( 1/3)-D-Gal-
b
a
identify and to study glycosyltransferases that catalyze the third
step of biosynthesis of O-antigenic polysaccharide repeating units
in Pseudomonas aeruginosa and E.coli O104 respectively.
Scheme 1. Reagents and conditions: a) cyclohexanone diethyl ketal, TsOH, MeCN, 20ꢀ
C, 4: 98%; b) cyclohexanone diethyl ketal, TsOH, MeCN, 20ꢀ C, 6: 45%.
D-Rhamnose polymers can be found in virtually all Pseudomonas
aeruginosa species but also in variety of other bacteria, e.g. Steno-
trophomonas maltophilia, Azospirillum brasilense, Azorhizobium
caulinodans, Pantoea agglomerans and E.coli O99. Thus, the synthetic
cyclohexylidene-2-deoxy-
conditions affording benzyl 2-acetamido-2-deoxy-4,6-O-cyclo-
hexylidene-3-O-(2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-
D-galactopyranoside 10 in a yield of 42% (Scheme 2).
After removal of the cyclohexylidene protecting group from 8
and 10 with 90% CF3COOH, diols 11(95%) and 12 (93.6%) were ob-
tained, and were further converted to derivatives 13 and 14 in a
yield of 98%.
Removing the benzyl group from 13 or 14 by hydrogenolysis
afforded 15 or 16 in a yield of 90%. The derivatives were treated
with bis(benzyloxy)(diisopropylamino)phosphine [10] in the
presence of tetrazole followed by oxidation with m-chlor-
a-D-galactopyranoside 6 under similar
b
a-
compound D-Rha(
serve as an adaptor structure for the extension of D-Rhamnose
oligosaccharides or polymers in many different bacteria. The Gal 1-
a1/3)-D-GlcNAca-PP-UnOPh 1 can potentially
b
3GalNAc-structure at reducing end is found in E.coli serotypes O5,
O22, O36, O69, O71, O107, O117, O155, O160, O171, O174, and O178.
In addition, it is found in other serotypes as internal structure. The
synthetic compound D-Gal(b1/3)-D-GalNAca-PP-UnOPh 2 can be
a substrate for many enzymes that extend the O-antigen repeating
unit chain. Toxicity is unknown but since the enzymatic studies are
done in vitro, the toxicity is not relevant.
operbenzoic acid (mCPBA), and dibenzyl a-glycosyl phosphates 17
or 18 were obtained in a yield of 85%. Hydrogenolysis of the latter
compounds in the presence of 10% Pd(OH)2/C in glacial acetic acid
2. Results and discussion
resulted in acetylated a-glycosyl dihydrogen phosphates 19 or 20 in
a yield of 90% (Scheme 3).
To synthesize the target disaccharide derivatives, the Helferich
method [8] (glycosylation of an alcohol using a glycosyl halide as
the glycosyl donor and a mercury cyanide as the promoter) was
used.
Activating 11-phenoxyundecyl phosphate 21 [2] with 1,1-
carbonyldiimidazole (CDI) for 2 h led to phosphoroimidazolidate
intermediate 22 (Rf 0.9, CHCl3/MeOH/H2O 6:4:0.5). Following the
condensation of crude 22 with acetylated glycosyl phosphates 19 or
20 at 37 ꢀC for 28 h resulted in protected diphosphates 23 or 24 (Rf
0.6). Small quantities of the symmetrical bis-(phenoxyundecyl)
diphosphate intermediate 22 (Rf 0.7), and acetylated sugar phos-
phates 19 or 20 (Rf 0.3) were also present in the reaction mixture.
Reaction mixtures containing 23 and 24 were deacetylated with
sodium methoxide in methanol followed by neutralization with
AcOH. The desired sodium salts of phenoxyundecyl diphosphate
disaccharides 1 or 2 were isolated by reversed-phase chromatog-
raphy on 5 mL C18 Sep-Pak cartridges in total yields of 60% for 1 and
of 59% for 2 (Scheme 4).
Benzyl 2-acetamido-4,6-O-cyclohexylidene-2-deoxy-
copyranoside 4 and benzyl 2-acetamido-4,6-O-cyclohexylidene-2-
deoxy- -D-galactopyranoside 6 were chosen as glycosyl acceptors
a-D-glu-
a
for the synthesis of the corresponding protected disaccharides
since they can easily be obtained by the interaction of benzyl 2-
acetamido-2-deoxy-
a
-D-glucopyranoside [12]
3 or benzyl 2-
acetamido-2-deoxy-
a
-D-galactopyranoside [12] 5 with cyclohexa-
none diethyl ketal in acetonitrile in the presence of catalytic
amounts of TsOH (Scheme 1) according to the procedure described
in Ref. [13]. A lower yield of 6 was apparently a result of the
competitive formation of the 3,4-O-cyclohexylidene derivative as a
by-product which was separated by column chromatography on
SiO2.
The structure of 1 was confirmed by NMR and mass spectrom-
etry data. The 1H NMR spectrum in CD3OD represented a super-
2,3,4-Tri-O-acetyl-
tained by reaction of 1,2,3,4-tetra-O-acetyl-
a
-D-rhamnopyranosyl bromide 7 was ob-
-D-rhamnopyranose
position of the (2-acetamido-2-deoxy-3-O-
-D-glucopyranosyl) phosphate and 11-phenoxyundecyl phos-
phate (21) spectra with the ratio of the components 1: 1. The 31P
NMR spectrum contained two doublets at
ꢁ9.9 (JP1,P2 20.2 Hz) and
a-D-rhamnopyranosyl-
a
a
[10] with 40% solution of HBr in glacial AcOH and without purifi-
cation was glycosylated with benzyl 2-acetamido-4,6-O-cyclo-
d
hexylidene-2-deoxy-
the presence of Hg(CN)2 affording benzyl 2-acetamido-4,6-O-
cyclohexylidene-2-deoxy-3-O-(2,3,4-tri-O-acetyl- -D-rhamnopyr-
anosyl)- -D-glucopyranoside 8 in a yield of 40%.
2,3,4,6-Tetra-O-acetyl- -D-galactopyranosyl bromide
obtained analogously from 1,2,3,4,6-penta-O-acetyl-
actopyranose and was condensed with benzyl 2-acetamido-4,6-O-
a-D-glucopyranoside 4 in dry acetonitrile in
d
ꢁ12.4 (JP2,P1 20.2 Hz) ppm, which are typical for non-symmetrical
P1,P2-diesters of pyrophosphoric acid. The HRESIMS spectrum
(negative ion mode) of 1 contained a signal m/z 772.2691, which
corresponds to [M e H]e for compound (1).
a
a
a
9
was
The structure of the compound 2 was also confirmed by NMR
b
-D-gal-
and MS data. The 1H NMR spectrum in CD3OD represented a

V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
21
Scheme 2. Reagents and conditions: a) CH3CN, Hg(CN)2, 8: 40%; b) CH3CN, Hg(CN)2, 10: 42%.
Scheme 3. Reagents and conditions: a) 90% CF3COOH, 11 (95%), 12 (42%); b) Ac2O/Py, 13 or 14: 98%; c) H2- Pd(OH)2/C, EtOH, 15 or 16:90%; d) i) (BnO)2PN(íPr)2, tetrazole, CH2Cl2; ii)
mCPBA, 17 or 18: 85%; e) H2-Pd(OH)2/C, AcOH, 19 or 20: 90%.
superposition of the (2-acetamido-2-deoxy-3-O-
anosyl- -D-galacopyranosyl) phosphate and 11-phenoxyundecyl
phosphate (21) spectra with the ratio of the components 1: 1. The
31P NMR spectrum contained two doublets at
ꢁ10.05 (JP1,P2
b
-D-galactopyr-
sialyltransferase WbwA from Escherichia coli serotype O104 [9].
a
3. Experimental
d
20.2 Hz) and
d
ꢁ12.4 (JP2,P1 20.2 Hz) ppm, which are typical for non-
3.1. General
symmetrical P1,P2-diesters of pyrophosphoric acid. The HRESIMS
spectrum (positive ion mode) of 2 contained a signal m/z 812.2618,
which corresponds to [M þ Na]þ for compound (2).
The reactions were performed with the use of commercial re-
agents (Aldrich, Fluka, Acros Organics). Concentration of organic
solutions was performed under reduced pressure below 40 ꢀC.
The target compounds 1 and 2 are currently examined as
acceptor substrates in biochemical assays. Compound 1 is the
adaptor compound for the extension of the сommon poly-
saccharide antigen by D-rhamnosyltransferases from Pseudomonas
Column chromatography was carried out on a silica gel (40e60 mm,
Acros Organics). Thin-layer chromatography was performed using
glass-based Silica Gel 60 F254 plates (Merck, Germany). The 1H, 13C,
and 31P NMR spectra were recorded for solutions in CDCl3 (in
aeruginosa [4]. Compound 2 is the acceptor substrate for
a2,3-

22
V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
Scheme 4. Reagents and conditions: a) i) íPr2NH, C6H6; ii) CDI/THF; iii) MeOH; b) íPr2NH, THF; c) i) MeONa/MeOH; ii) AcOH; iii) C18 Sep-Pak/water-MeOH, sodium salt 23: total
yield 60%; sodium salt 24: total yield 59%; A and B see Scheme 3.
CD3OD for compounds 1 and 2) on a Bruker AM-300 instrument
(300.13 MHz for 1H) or on a Bruker AVANCE 600 spectrometer (for
1H, 600.13, for 13C, 150.90, and for 31P, 242.9 MHz). Chemical shifts
C
C
C
21H29NO6: 391.1995; m/z ¼ 414.1873 [M þ Na]þ, calcd for
21H29NO6Na: 414.1887; m/z ¼ 430.1615 [M þ K]þ, calcd for
1
21H29NO6K: 430.1626; H NMR:
d 7.50e7.20 (m, 5H, CH2Ph); 5.80
(
d
) are reported in parts per million (ppm), and signals are
(d, 1H, J 9 Hz, NH); 5.00 (d, 1H, J 4 Hz, H-1); 4.72e4.50 (m, 2H,
PhCH2O); 4.50e4.40 (m, 1H, H-5); 4.20 (d,1H, J 2 Hz, H-4);
4.10e3.70 (m, 4H, H-6, H-6 0, H-3, H-2); 2.70 (s, 1H, OH); 2.00 (s, 3H,
NHCOCH3); 2.00e1.30 (m, 10H, C6H10).
described as s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), and m (multiplet). 1H NMR chemical shifts were refer-
enced to residual signal of CHCl3 (dH 7.27) or CD2HOD (dH 3.33). 13
C
chemical shifts were referenced to the central resonance of CDCl3
dC 77.0) and CD3OD (dC 49.0). Phosphorus chemical shifts are given
relative to those of 85% phosphoric acid:
the signals in the NMR spectra were confirmed using 2D-spec-
troscopy (COSY, HSQC, HMBC). High-resolution mass spectra
(electrospray ionization, HRESIMS) were recorded in positive mode
(negative mode for 1) on a Bruker micrOTOF II mass spectrometer
for 2 ꢂ 10ꢁ5 M solutions in MeCN or MeOH. Optical rotations were
measured using a JASCO P-2000 automatic digital polarimeter
(
d
¼ 0 ppm. Assignments of
3.4. Benzyl 2-acetamido-4,6-O-cyclohexylidene-2-deoxy-3-O-
(2,3,4-tri-O-acetyl-a-D-rhamnopyranosyl)-a-D-glucopyranoside
(8)
1,2,3,4-Tetra-O-acetyl-a-D-rhamnopyranose [10] (500 mg,
1.5 mmol) was dissolved in glacial AcOH (1 mL), cooled to 0 ꢀC, and
5 mL of a 40% solution of HBr in glacial AcOH was added with
stirring. After 2 h at rt TLC (toluene/acetone 4:1) showed the
presence of one chromatographic zone (Rf 0.5) corresponding to
bromide 7. The reaction mixture was freeze-dried. The suspension
of 4 (390 mg, 1 mmol), Hg(CN)2 (500 mg, 2 mmol), molecular sieves
3 Å (500 mg, powder) in dry acetonitrile (2 mL) was stirred for
30 min at rt, a solution of 7 in dry acetonitrile (2 mL) was added
dropwise and stirring was continued overnight. The reaction
mixture was diluted with CHCl3 (50 mL), filtered and washed
consecutively with sat. aq. KI (50 mL) and H2O (2 ꢂ 25 mL). The
organic phase was dried with MgSO4, and the solvent was evapo-
rated. The residue was subjected to column chromatography
(Japan) for solutions in CDCl3 with concentration c ¼ 1 unless stated
20
otherwise. [
a
]
values are given in units of 10ꢁ1deg cm3gꢁ1
.
D
Reverse-phase chromatography was conducted with C18 cartridges
(Sep-Pak, Waters, United States, 5 mL).
3.2. Benzyl 2-acetamido-4,6-O-cyclohexylidene-2-deoxy-a-D-
glucopyranoside (4)
Compound 4 was obtained from benzyl 2-acetamido-2-deoxy-
a
-D-glucopyranoside [12] (3) according to the procedure described
20
[13] in a yield of 98%. Rf 0.3 (toluene/acetone 1: 1); [
a]
þ100.3;
D
HRESIMS: m/z ¼ 392.2055 [M þ H]þ, calcd for C21H29NO6:
(toluene/acetone 1: 0 / 1: 1) affording compound 8 as a foam
Na]þ, calcd for
20
(265 mg, 40%): Rf 0.5 (toluene/acetone 2:1); [a]
þ80.6ꢀ; HRE-
391.1995; m/z
C
¼
414.1870 [M
þ
D
21H29NO6Na:414.1887; 1H NMR:
d 7.20e7.50 (m, 5H, CH2Ph); 5.90
SIMS: m/z ¼ 664.2950 [MþH]þ, calcd for C33H45NO13: 663.2891; m/
z ¼ 686.2770 [MþNa]þ, calcd for C33H45NO13Na 686.2783; 1H NMR
(d, 1H, J 9 Hz, NH); 4.90 (d, 1H, J 4 Hz, H-1); 4.75e4.40 (m, 2H,
PhCH2O); 4.20 (m, 1H, H-5); 3.90e3.60 (m, 5H, H-6, H-6 0, H-4, H-3,
H-2); 3.00 (s, 1H, OH); 2.00 (s, 3H, NHCOCH3); 2.00e1.30 (m, 10H,
C6H10).
(D-Rha ¼ II; GlcNAc ¼ I):
d 7.40e7.30 (m, 5Н, СН2Рh); 5.64 (d, 1Н, J
9.6 Hz, NH); 5.39 (br s, 1Н, H-2II); 5.19 (dd, 1Н, J3,2 3.6 Hz, J3,4 9.6 Hz,
H-3II); 5.02 (br s, 1Н, H-1II), 5.02 (t, 1Н, J4,3 9.6 Hz, J4,5 9.6 Hz, H-4II);
4.87 (d, 1Н, J1,2 3.6 Hz, H-1I); 4.71e4.44 (m, 2H, OCH2Ph); 4.33e4.30
(m, 1Н, H-2I); 3.92e3.90 (m, 1Н, H-5II); 3.81e3.80 (m, 5Н, H-
3I,4I,5I,6I,60I); 2.12 (s, 3H, OAc); 2.04 (s, 3H, OAc); 1.95 (s, 3H, OAc);
1.94 (s, 3H, NHAc); 2.00e1.30 (m,10Н, C6H10): 1.19 (d, 3H, J6,5 6.6 Hz,
3.3. Benzyl 2-acetamido-4,6-O-cyclohexylidene-2-deoxy-a-D-
galactopyranoside (6)
Compound
6
was synthesized from benzyl 2-deoxy-2-
H-6II); 13С NMR: 100.1 (quaternary carbon atom), 99.3 (C-1II); 97.4
d
acetamido- -D-galactopyranoside [12] (5) according to the pro-
a
(C-1I); 76.8 (C-3I); 73.9 (C-5I); 70.8 (C-4II); 69.8 (CH2Ph); 69.6 (C-
2II); 69.1 (C-3II); 66.7 (C-5II); 63.8 (C-4I); 61.5 (C-6I); 51.7 (C-2I); 23.1
(NHCOCH3); 20.7 (OCOCH3); 17.5 (C-6II).
cedure described [13] in a yield of 45%. Rf 0.3 (toluene/acetone 1:
20
1); [
a
]
þ120.6; HRESIMS: m/z ¼ 392.2056 [M þ H]þ, calcd for
D

V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
23
20
3.5. Benzyl 2-acetamido-2-deoxy - 3-O-(2,3,4-tri-O-acetyl-
a
-D-
1 / 0: 1) affording 17 (220 mg, 85%): Rf 0.5 (EtOAc); [
a]
þ44.4;
D
rhamnopyranosy)- -D-glucopyranoside (11)
a
HRESIMS: m/z
¼
838.2658 [MþH]þ, calcd for C38H48NO18Р:
837.2609; m/z ¼ 860.2488 [MþNa]þ, calcd for C38H48NO18РNa:
Protected disaccharide 8 (250 mg, 0.38 mmol) was dissolved in
90% aqueous CF3COOH (1 mL) and the reaction mixture was stirred
at rt. After 15 min TLC (toluene/acetone 2: 1) showed complete
reaction. Ethanol (10 mL) and toluene (10 mL) were added, and the
reaction mixture was evaporated to dryness. The syrupy residue
was subjected to column chromatography with linear gradient
(toluene/acetone) affording 11 as a foam (208 mg, 95%): Rf 0.5
(toluene/acetone 1: 1).
860.2501; 1H NMR (Rha ¼ II, GlcNAc ¼ I):
d 7.40e7.30 (m, 5H,
СH2Рh); 5.69 (dd, 1H, J1,2 3.0 Hz, J1,Р 5.4 Hz, H-1I); 5.64 (d, 1H, J 9 Hz,
NH); 5.15e5.07 (m, 8H, H-2II, 3II, 4II, 4I, OСH2Ph); 4.74 (br s, 1H, H-
1II); 4.33e4.30 (m,1H, H-2I); 4.10e4.07 (m,1H, H-6I); 3.93e3.85 (m,
3H, H-5II, 5I, 60I); 3.67 (t,1H, J3,4 10.2 Hz, J2,3 10.2 Hz, H-3I); 2.15e1.75
(m, 18H, COCH3, NHCOCH3); 1.16 (d, 3H, J6,5 6.6 Hz, H-6II); 13С NMR:
d
100.5 (C-1II); 96.6 (d, J1,Р 6.8 Hz, C-1I); 79.0 (C-3I); 61.5 (C-6I); 52.2
(d, J2,Р 7.1 Hz, C-2I); 23.0 (NHCOCH3); 20.5 (ОCOCH3); 17.6 (C-6II);
31Р NMR: ꢁ2.4 (s).
3.6. Benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4-tri-O-
acetyl-
a-D-rhamnopyranosyl)-
a-D-glucopyranoside (13)
3.9. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-
a-D-rhamnopyranosyl)-a-D-glucopyranosyl dihydrogen
A solution of 11 (208 mg, 0.316 mmol) in a mixture of dry pyr-
idine (1 mL) and Ac2O (1 mL) was stirred overnight at room tem-
perature. Ethanol (10 mL) and toluene (10 mL) were added and the
reaction mixture was evaporated to dryness. The syrupy residue
was subjected to column chromatography (CHCl3 / acetone),
phosphate (19)
A solution of 17 (130 mg, 0.155 mmol) in glacial AcOH (5 mL)
was hydrogenated in the presence of 10% Pd(OH)2/C (50 mg) at
36 ꢀC with shaking until the benzyl groups were completely
removed (TLC: EtOAc). The catalyst was separated by centrifugation
at 10,000 rpm and the supernatant was freeze-dried. Removal of
benzyl group decreases Rf (EtOAc) value of compound to 0, the
starting material disappears affording compound 15 as a white
powder (180 mg, 90%): Rf 0; HRESIMS: m/z ¼ 658.1755 [MþH]þ,
calcd for C24H36NO18Р: 657.1670; m/z ¼ 680.1567 [MþNa]þ, calcd
for C24H36NO18РNaþ: 680.1562; 31Р NMR:ꢁ1.4 (br s). The product
was used at the next step of the synthesis without additional
purification.
affording 13 as a foam (235 mg, 98%): Rf 0.7 (CHCl3/acetone 4: 1);
20
[
a
]
þ160.1; HRESIMS: m/z
¼
668.2550 [MþH]þ, calcd for
D
C
C
d
31H41NO15
:
667.2476; m/z
¼
690.2349 [MþNa]þ, calcd for
31H41NO15Na:690.2368; 1H NMR (Rha
¼
II, GlcNAc
¼
I):
7.40e7.30 (m, 5Н, СН2Рh); 5.63 (d, 1Н, J 9 Hz, NH); 5.16 (dd, 1Н, J3,2
3 Hz, J3,4 9.6 Hz, H-3II); 5.12 (dd,1H, J4,3 9.6 Hz, J4,5 9.6 Hz, H-4I); 5.03
(br s, 1H, H-2II); 5.02 (t, 1H, J4,3 10.2 Hz, J4,5 10.2 Hz, H-4II); 4.97 (d,
1H, J1,2 3.6 Hz, H-1I); 4.78 (br s, 1H, H-1II); 4.69e4.47 (m, 2H,
OCH2Ph); 4.34e4.30 (m, 1H, H-2I); 4.20e4.17 (m, 2H, H-6I),
3.93e3.88 (m, 2H, H-5II, H-5I); 3.88 (t, 1H, J3,4 9.6 Hz, J3,2 9.6 Hz, H-
3I); 2.14e1.90 (m, 18H, OCOCH3, NHCOCH3); 1.17 (d, 3H, J6,5 6.6 Hz,
3.10. Sodium P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-
3-O-a-D-rhamnopyranosyl-a-D-glucopyranosyl) diphosphate (1)
H-6II); 13С NMR:
d
99.9 (C-1II); 96.7 (C-1I); 80.4 (C-3I); 70.8 (C-4II);
70.1 (C-2II); 70.1 (CH2Ph); 70.0 (C-4I); 68.6 (C-3II); 68.3 (C-5I); 67.2
(C-5II); 62.1 (C-6I); 52.2 (C-2I); 23.4 (NHCOCH3); 20.6 (ОCOCH3);
17.7 (C-6II).
Dry diisopropylamine (0.3 mL) was added to a solution of 11-
phenoxyundecyl dihydrogen phosphate [2] (21, 76 mg,
0.22 mmol) in dry benzene (4 mL). The mixture was stirred under
argon atmosphere for 5 min and then freeze-dried. The resulting
white powder was dissolved in dry THF (6 mL), and CDI (270 mg,
1.66 mmol) was added. The reaction mixture was stirred at rt under
argon atmosphere. After 2 h TLC (CHCl3/MeOH/H2O 6:4:0.5)
showed total conversion of 21 (Rf 0.5) into intermediate phos-
phoroimidazolidate 22 (Rf 0.85). Dry MeOH (1 mL) was added and
the mixture was stirred for 30 min at rt. The solvent was removed in
vacuum, the residue was dissolved in dry benzene (6 mL) and
freeze-dried affording crude 11-phenoxyundecyl phosphor-
oimidazolidate 22.
A mixture of glycosyl dihydrogen phosphate 19 (92 mg,
0.14 mmol), dry benzene (6 mL) and dry diisopropylamine (0.3 mL)
was stirred under argon atmosphere and freeze-dried. Solution of
crude phosphoroimidazolidate 22 in dry THF (3 mL) was added to
the lyophilizate with stirring under argon atmosphere. The reaction
mixture was left at 37 ꢀC for 48 h, the solvent was evaporated under
reduced pressure, the oily residue was dissolved in MeOH (3 mL)
3.7. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4-tri-O-acetyl-
a-D-rhamnopyranosyl)-a-D-glucopyranose (15)
A solution of 13 (235 mg, 0.35 mmol) in ethanol (10 mL) was
hydrogenated in the presence of 10% Pd(OH)2/C (50 mg) at 36 ꢀC
with stirring until the benzyl group was completely removed (TLC:
CHCl3/acetone 8: 2). The catalyst was separated by centrifugation at
10,000 rpm, and the supernatant was evaporated to dryness
affording compound 15 (180 mg, 90%): Rf 0.1 (CHCl3/acetone 4: 1);
20
[
C
C
a
]
þ40.5; HRESIMS: m/z
¼
578.2079 [MþH]þ, calcd for
D
24H35NO15
:
577.2007; m/z
¼
600.1898 [MþNa]þ, calcd for
24H35NO15Na: 600.1899. The product was used in the next step of
the synthesis without purification.
3.8. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4-tri-O-acetyl-
a-D-rhamnopyranosyl)-
a-D-glucopyranosyl dibenzyl phosphate
(17)
and 1 M solution of MeONa in dry methanol (200 mL) was added.
To a stirred solution of 15 (180 mg, 0.31 mmol) and tetrazole
(230 mg, 3.0 mmol) in dry CH2Cl2 (5 mL) under an argon atmo-
sphere and ꢁ60 ꢀC bis (benzyloxy)(diisopropylamino)phosphine
[11] (640 mg, 2 mmol) was added dropwise with stirring. The so-
lution was allowed to warm to rt and stirred for 5 h under argon
atmosphere. The reaction mixture was cooled to ꢁ60 ꢀC, mCPBA
(400 mg, 2 mmol) was added with stirring followed by warming to
rt. After 18 h of stirring the solution was diluted with CHCl3
(100 mL) and washed with sat. aq. Na2S2O3 (50 mL), sat. aq.
NaHCO3, and water. The organic phase was concentrated. The res-
idue was subjected to column chromatography (toluene/EtOAc 1:
After stirring for 1 h at rt glacial AcOH (0.6 mL) was added, the
solution was stirred for 15 min and evaporated to dryness in vac-
uum. The solid residue was dissolved in water (40 mL), applied on
C18 Sep-Pak cartridge preactivated with EtOH and elution was
carried out from water (40 mL) to 5, 10, 15, 20 and 30% v/v solution
of EtOH in water (20 mL of each); the desired product was eluted
with 10 and 15% solution of EtOH in water. The combined fractions
were evaporated under reduced pressure, the residue was dis-
solved in 5 mL of water, and the solution was freeze-dried affording
1 as a white powder (68 mg, 60%): Rf 0.65 (CH3Cl/MeOH/H2O
20
10:10:3); [
a]
þ62.0; HRESIMS: m/z ¼ 772.2691 [M-H]-, calcd for
D

24
V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
C31H52NO17Р2: 772.2716; 1H NMR (Rha ¼ II, GlcNAc ¼ I):
d
8.85 (s,
4.2 Hz, H-1I); 4.95 (dd, 1H, J3,4 3.6 Hz, J2,3 10.2 Hz, H-3II); 4.58 (d, 1H,
J1,2 8.4 Hz, H-1II); 4.55e4.51 (m, 1H, H-2I); 4.20e4.10 (m, 3H, H-5I,
H-60I, H-6II); 4.04e4.01 (m, 1H, H-6I); 4.00 (dd, 1H, J3,4 3.0 Hz, J3,2
11.4 Hz, H-3I); 3.86e3.85 (m, 1H, H-5II); 2.14e1.96 (m, 21H, COCH3,
1H, H-2 imidazole salt); 7.52 (s, 2H, H4, H-5 imidazole salt)
7.25e7.22 (m, 2H, PhO); 6.90e6.85 (m, 3H, PhO); 5.51 (dd, 1H, J1,2
3.6 Hz, J1,Р 6.6 Hz, H-1I); 5.13 (bs, 1H, H-1II); 4.10e4.05 (m, 1H, H-2I);
4.02e3.98 (m, 2H, CH2OP); 4.00e3.90 (m, 4H, H-2II, 5I, PhOCH2);
3.83e3.76 (m, 2H, H-6I, 3I); 3.70e3.60 (m, 3H, H-60I, 3II, 5II); 3.46 (t,
1H, J4,3 10.2 Hz, J4,5 10.2 Hz, H-4I); 3.34 (t,1H, J4,3 10.2 Hz, J4,5 10.2 Hz,
H-4II), 2.05 (s, 3H, NHCOCH3), 1.80e1.30 (m, 18H, СH2); 1.27 (d, 3H,
NHCOCH3); 13С NMR: 100.5 (C-1II), 97.2 (C-1I), 72.8 (C-3I), 71.0 (C-
d
5I), 70.9 (C-3II), 70.8 (CH2Ph), 68.8 (C-4I), 68.5 (C-2II), 67.6 (C-5I),
66.8 (C-4II), 62.9 (C-6I), 61.1 (C-6II), 48.5 (C-2I), 23.3 (NHCOCH3),
20.5 (ОCOCH3).
J5,6 6 Hz, H-6II); 13С NMR:
d 174.1 (CO); 135.7 (C-2, imidazolium
salt); 130.5 (C-4, C-5, imidazolium salt); 121.6, 120.6, 115.7 (PhO);
103.2 (C-1II); 96.60 (d, J1,Р 6.3 Hz, C-1I); 79.8 (C-3I); 75.0 (C-5I); 74.2
(C-4II); 72.9 (C-4I); 72.6 (C-3II); 72.4 (C-2II); 70.3 (C-5II); 69.1
(PhOCH2); 67.4 (d, JP,C 6 Hz, CH2OP); 62.6 (C-6I); 53.9 (d, J2,P 8.5 Hz,
C-2I); 32.0 (d, JC,P2 5.8 Hz, CH2CH2OP); 30.85, 30.83, 30.39,30.64,
30.58, 27.31, 27.04 (CH2); 23.4 (NHCOCH3), 18.3 (C-6II); 31Р NMR:
3.14. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-galactopyranosyl)-a-D-galactopyranose (16)
Compound 14 (200 mg, 0.28 mmol) was hydrogenated as
described for synthesis of 15 affording compound 16 (160 mg, 90%):
Rf 0.05 (toluene/EtOAc 2:1); [
a
]
20 þ45.3; HRESIMS: m/z ¼ 636.2143
D
d
ꢁ9.9 (d, JP2,P1 20.2 Hz, P-2); ꢁ12.4 (d, JP1,P2 20.2 Hz, P-1).
[MþH]þ, calcd for C26H37NO17: 635.2061; m/z
¼
658.1963
[MþNa]þ, calcd for C26H37NO17Na: 658.1959. The compound was
used without further purification.
3.11. Benzyl 2-acetamido-4,6-O-cyclohexylidene-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-a-D-
galactopyranoside (10)
3.15. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-
b-D-galactopyranosyl)-a-D-galactopyranosyl dibenzyl
A mixture of 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bro-
phosphate(18)
mide (9) [14] (620 mg, 1.5 mmol), 4 (490 mg, 1.25 mmol), Hg(CN)2
(311 mg,1.25 mmol), molecular sieves 3 Å (100 mg) and acetonitrile
(15 mL) was stirred overnight at room temperature. The reaction
mixture was diluted with CHCl3 (50 mL), the precipitate was
filtered off and the filtrate was washed with sat. aq. KI (50 mL) and
H2O (2 ꢂ 25 ml). The organic phase was dried (MgSO4) and the
solvent was evaporated. The residue was subjected to column
Treatment of 16 (95 mg, 0.15 mmol) with bis(benzylox-
y)(diisopropylamino)phosphine [11] (320 mg, 1 mmol) was con-
ducted analogously to the synthesis of 17 affording compound 18 as
20
a foam (115 mg, 85%): Rf 0.5 (EtOAc), [
a]
þ53.3; HRESIMS: m/
D
z ¼ 896.2739 [MþH]þ, calcd for C40H50NO20Р: 895.2664; m/
z ¼ 918.2565 [MþNa]þ, calcd for C40H50NO20РNa: 918.2561; 1H
chromatography (toluene/EtOAc) affording 10 as a foam (380 mg,
NMR (Gal ¼ A II, GalNAc ¼ B I):
d 7.40e7.30 (m, 5H, СH2Рh); 5.79 (d,
20
42%): Rf 0.7 (toluene/EtOAc 2:1); [
a
]
D
þ88.2; HRESIMS: m/
1H, J 9.0 Hz, NH); 5.75 (dd, 1H, J1,2 3.6 Hz, J1,Р 6.0 Hz, H-1I); 5.40 (br
z
¼
722.3018 [MþH]þ, calcd for C35H47NO15
:
721.2946; m/
d, 1H, J3,4 3.0 Hz, H-4I), 5.36 (br d, 1H, J3,4 3,6 Hz, H-4II); 5.10 (dd, 1H,
z ¼ 744.2836 [MþNa]þ, calcd for C35H47NO15Naþ:744.2843, m/
J
1,2 8.4 Hz, J2,3 10.2 Hz, H-2II); 5.09e5.01 (m, 4H, OCH2Ph); 4.96 (dd,
z ¼ 760.2577 [MþК]þ, calcd for C35H47NO15Kþ:760.2583; 1H NMR
1H, J3,4 3.6 Hz, J2,3 10.2 Hz, H-3II), 4.58 (d, 1H, J1,2 8.4 Hz, H-1II);
4.57e4.52 (m, 1H, H-2I), 4.25 (t, 1H, H-5I); 4.16e4.09 (m, 4H, H-6II,
6I); 3.93e3.88 (m, 2H, H-5II,3I); 2.15e1.80 (m, 21H, OCOCH3,
(Gal ¼ II, GalNAc ¼ I):
d 7.50e7.20 (m, 5H, СH2Рh); 5.55 (d, 1H, J
9 Hz, NH); 5.36 (d, 1H, J3,4 3.6 Hz, H-4II); 5.15 (dd, 1H, J1,2 8.4 Hz, J2,3
10.2 Hz, H-2II); 5.10 (d, 1H, J1,2 4 Hz, H-1I); 4.98 (dd, 1H, J2,3 10.2, J3,4
3.6 Hz, H-3II); 4.71 (d, 1H, J1,2 8.4 Hz, H-1II); 4.71e4.50 (m, 2H,
OCH2Ph); 4.27 (bd, 1H, J3,4 3 Hz, H-4I); 3.90e3.80 (m, 2H, H-3II, 5II);
2.20e1.90 (m, 15H, OCOCH3, NHCOCH3), 2.00e1.30 (m, 10H, C6H10).
NHCOCH3); 13С NMR:
d 170.5e169.6; 128.7e128,2 (aromatic); 100.5
(C-1II); 97.5 (d, J1,P 6.8 Hz, C-1I); 71.6 (C-3I); 71.0 (C-5II); 70.8 (C-3II);
66.8 (C-4II); 62.1 (C-6I); 61.2 (C-6II); 48.7 (d, JP,27Hz, C-2I); 22.8
(NHCOCH3); 20.5 (ОCOCH3); 31Р NMR: ꢁ2.4 (s).
3.12. Benzyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-
D-galactopyranosyl)- -D-galactopyranoside (12)
b
-
3.16. 2-Acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-galactopyranosyl)-a-D-galactopyranosyl phosphate (20)
a
Compound 10 (300 mg, 0.42 mmol) was treated with 90%
A solution of 18 (50 mg, 0.056 mmol) in glacial AcOH (5 mL) was
hydrogenated with 10% Pd(OH)2/C (10 mg) as described for 17
affording dihydrogen phosphate 20 as a white powder (36 mg,
90%): Rf 0 (EtOAc); 31Р NMR: ꢁ1.5 (br s). The compound was used
without further purification.
CF3COOH as described for synthesis of 11 affording diol 12 as a foam
20
(250 mg, 93.6%): Rf 0.7 (toluene/ethyl acetate 2:1); [
a
]
þ65.2;
D
HRESIMS: m/z
¼
642.2398 [MþH]þ, calcd for C29H39NO15
:
641.2320; m/z ¼ 664.2225 [MþNa]þ, calcd for C29H39NO15Na:
664.2217.
3.17. Sodium P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-
3.13. Benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-
O-b-D-galactopyranosyl-a-D-galactopyranosyl) diphosphate (2)
tetra-O-acetyl-b-D-galactopyranosyl)-a-D-galactopyranoside (14)
Diphosphate
described for the synthesis of diphosphate
2
was prepared similarly to the procedure
from 11-
A solution of 12 (200 mg, 0.31 mmol) in a mixture of dry pyri-
dine (1 mL) and Ac2O (1 mL) was stirred overnight at rt, ethanol
(10 mL) and toluene (10 mL) was added and the reaction mixture
was evaporated to dryness. The syrupy residue was subjected to
1
phenoxyundecyl dihydrogen phosphate [2] (28 mg, 0.08 mmol),
CDI (100 mg, 0.61 mmol) and freshly made glycosyl dihydrogen
phosphate 20 (36 mg, 0.05 mmol). The target compound was eluted
from C18 Sep-Pak cartridge with 10, 15 and 20% of EtOH. Fractions
containing the target compound were concentrated and freeze-
column chromatography (toluene/acetone) affording 14 as a foam
20
(220 mg, 98%): Rf 0.5 (toluene/EtOAc 2:1); [
a]
þ85.3; HRESIMS:
D
m/z ¼ 726.2613 [MþH]þ, calcd for C33H43NO17: 725.2531; m/
dried affording 2 as a white powder (25 mg, 59%): Rf 0.6 (CH3Cl/
z ¼ 748.2425 [MþNa]þ, calcd for C33H43NO17Na: 748.2429; 1H NMR
MeOH/H2O 10:10:3), [
a]
þ68.5; HRESIMS: m/z ¼ 812.2618
20
D
(Gal ¼ II, GalNAc ¼ I):
d
7.40e7.30 (m, 5H, СH2Рh); 5.64 (d, 1H, J
[MþNa]þ, calcd for C31H52NNaO18Р2: 811.2557; m/z ¼ 834.2448
9.0 Hz, NH); 5.38 (bd, 1H, J3,4 3.0 Hz, H-4I); 5.34 (bd, 1H, J3,4 3,6 Hz,
H-4II); 5.12 (dd, 1H, J2,1 8.40 Hz, J2,3 10.2 Hz, H-2II); 5.04 (d, 1H, J1,2
[Mþ2Na]þ, calcd for C31H52NNa2O18Р2: 834.2450; 1H NMR (Gal ¼ II,
GalNAc ¼ I):
d 8.83 (s, 1H, H-2 imidazole salt); 7.51 (s, 2H, H4, H-5

V. Torgov et al. / Carbohydrate Research 453-454 (2017) 19e25
25
V.V. Veselovsky, I. Brockhausen, Biosynthesis of the common polysaccharide
antigen of Pseudomonas aeruginosa PAO1: characterization and role of WbpZ
imidazole salt) 7.25e7.22 (m, 2H, PhO); 6.89e6.87 (m, 3H, PhO);
5.60 (dd, 1H, J1,2 3.6 Hz, J1,Р 6.6 Hz, H-1I); 4.55e4.52 (m, 1H, H-2I);
4.42 (d, 1H, J1,2 7.2 Hz, H-1II); 4.20e4.16 (m, 1H, H-5I); 4.17 (br d, 1H,
e a GDP-d-rhamnose: GlcNAc/GalNAc-diphosphate-lipid
transferase WbpZ, J. Bacteriol. 197 (2015) 2012e2019.
a1,3-D-rhamnosyl-
J
J
3,4 2.4 Hz, H-4I); 3.99e3.94 (m, 4H, CH2OP, PhOCH2), 3.85 (br d, 1H,
3,4 3,0 Hz, H-4II); 3.55e3.54 (m, 1H, H-5II); 2.03 (s, 3H, NHCOCH3);
[5] S. Wang, D. Czuchry, B. Liu, A. Vinnikova, Y. Gao, J.Z. Vlahakis, W.A. Szarek,
L. Feng, L. Wang, I. Brockhausen, Characterization of two UDP-Gal: GalNAc-
diphosphate-lipid
b1,3-galactosyltransferases WbwC from Escherichia coli
1.78e1.28 (m, 18H, CH2); 13С NMR:
d 135.8 (C-2, imidazolium salt);
serotypes O104 and O5, J. Bacteriol. 196 (2014) 3122e3133.
[6] N.S. Utkina, L.L. Danilov, V.V. Veselovsky, V.I. Torgov, T.N. Druzhinina, Syn-
130.5 (C-4, C-5, imidazolium salt); 121.6, 120.7, 115.7 (PhO); 106.7
(C-1II), 96.9 (d, J1,P 5.9 Hz, C-1I); 79.3 (C-3I); 76.8 (C-5II); 74.9 (C-5I);
73.5 (C-2II); 70.5, 70.4 (C-4II, 4I); 69.0 (PhOCH2); 67.4 (d, J 6.2 Hz,
CH2OP); 63.0 (C-6I); 62.6 (C-6II); 50.2 (d, J2,P 7.8 Hz, C-2I); 32.0; 30.9;
30.8; 30.7; 30.5; 27.3; 27.1; 19.6 (CH2); 22.8 (NHCOCH3); 31Р NMR:
thesis of P1-(11-phenoxyundecyl)-P2-(
a
-D-galactopyranosyl)diphosphate and
P1-(11- phenoxyundecyl)-P2-(
a-D-glucopyranosyl)diphosphate and investi-
gation of their acceptor properties in the reaction of mannosyl residue
transfer catalyzed by mannosyltransferase from Salmonella nawport, Russ. J.
Bioorg. Chem. 4 (2012) 412e416.
[7] Y. Gao, B. Liu, S. Strum, J.S. Schutzbach, T.N. Druzhinina, N.S. Utkina,
V.I. Torgov, L.L. Danilov, V.V. Veselovsky, J.Z. Vlahakis, W.A. Szarek, L. Wang,
d
ꢁ10.1 (d, JP1,P2 20.2 Hz, P-2), ꢁ12.4 (d, JP1,P2 20.2 Hz, P-1).
I. Brockhausen, Biochemical characterization of WbdN,
a
b
1,3-
glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic
Escherichia coli O157, Glycobiology 22 (2012) 1092e1102.
Acknowledgments
[8] B. Helferich, J. Zirner, Zur Synthese von Tetraacetyl-hexosen mit freiem 2-
Hydroxyl. Synthese einiger Disaccharide, Chem. Ber. 95 (1962) 2604e2611.
[9] D. Czuchry, P. Desormeaux, M. Stuart, D. Jarvis, K.L. Matta, W.A. Szarek,
I. Brockhausen, Identification and biochemical characterization of the novel
This work was supported by the Russian Foundation for Basic
Research (Project 16-04-00372).
a
2,3-sialyltransferase WbwA from pathogenic Escherichia coli serotype O104,
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