Convenient synthesis and purification of N-trifluoroacetylated neuraminic acid tetraol: A key precursor for the synthesis of new sialyl donors containing labile O-acyl protecting groups

Article abstract of DOI:10.1080/07328303.2014.913058

A convenient synthesis and separation of α- and β-anomers of methyl (phenyl 3,5-dideoxy-2-thio-5-trifluoroacetamido-D-glycero-D-galacto- nonulopyranosid)onate (6a and 6b) on a multigram scale was developed. Both α- and β-isomers of 6 were obtained as crys

Full text of DOI:10.1080/07328303.2014.913058

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Convenient Synthesis and Purification
of N-Trifluoroacetylated Neuraminic
Acid Tetraol: A Key Precursor for
the Synthesis of New Sialyl Donors
Containing Labile O-Acyl Protecting
Groups
a
a
a
Nikita M. Podvalnyy , Alexander I. Zinin , Nelly N. Malysheva ,
a
a
Roman A. Kunetskiy & Leonid O. Kononov
a
N. D. Zelinsky Institute of Organic Chemistry of the Russian
Academy of Sciences, Moscow 119991, Russian Federation
Published online: 22 May 2014.
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To cite this article: Nikita M. Podvalnyy, Alexander I. Zinin, Nelly N. Malysheva, Roman A. Kunetskiy
Leonid O. Kononov (2014) Convenient Synthesis and Purification of N-Trifluoroacetylated
&
Neuraminic Acid Tetraol: A Key Precursor for the Synthesis of New Sialyl Donors Containing
Labile O-Acyl Protecting Groups, Journal of Carbohydrate Chemistry, 33:5, 213-224, DOI:
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Journal of Carbohydrate Chemistry, 33:213–224, 2014
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2014.913058
Convenient Synthesis
and Purification
of N-Trifluoroacetylated
Neuraminic Acid Tetraol: A Key
Precursor for the Synthesis
of New Sialyl Donors
Containing Labile O-Acyl
Protecting Groups
Nikita M. Podvalnyy, Alexander I. Zinin, Nelly N. Malysheva,
Roman A. Kunetskiy, and Leonid O. Kononov
N. D. Zelinsky Institute of Organic Chemistry of the Russian Academy of Sciences,
Moscow 119991, Russian Federation
A convenient synthesis and separation of α- and β-anomers of methyl (phenyl 3,
5
-dideoxy-2-thio-5-trifluoroacetamido-D-glycero-D-galacto-nonulopyranosid)onate (6a
and 6b) on a multigram scale was developed. Both α- and β-isomers of 6 were ob-
tained as crystals suitable for safe storage. The β-isomer forms a crystalline solvate
with methanol. Fully O-trichloroacetylated and O-trifluoroacetylated N-trifluoroacetyl
thiosialosides were synthesized in an efficient manner from the β-tetraol 6b.
Keywords Sialic acid; Trifluoroacetamide; Crystal solvate; O-Trifluoroacetylation
INTRODUCTION
Biological significance of α-glycosides of sialic acids[1–3] stimulates continuous
search of new approaches for their synthesis (for the reviews see refs. [4–6]).
Received February 19, 2014; accepted April 4, 2014.
Address correspondence to Leonid O. Kononov, N. D. Zelinsky Institute of Organic
Chemistry of the Russian Academy of Sciences, Leninsky prosp., 47, 119991, Moscow,
Russian Federation. E-mail: kononov@ioc.ac.ru; leonid.kononov@gmail.com
2
13

14 N. M. Podvalnyy et al.
2
Since both naturally occurring N-acetyl- and N-glycolyl-substituted sialo-
oligosaccharides are required for biological studies, the use of sialyl donors
[
7,8]
with a suitable temporary protection at N(5)
(e.g., trichloroethoxycarbonyl
[
Troc][
9–13]
and trifluoroacetyl [TFA] group
[14–21]
) is considered reasonable.
Moreover, N-TFA is not only a convenient protecting group.[ The task of
7,8]
the construction of a naturally occurring equatorial sialic bond is not a simple
one. Although various approaches to stereoselective α-sialylation have been
[
4–6]
suggested,
the efficient introduction of sialic acid residues into oligosac-
[
7–16]
[17,22]
charides remains problematic. Considerable influence of N
- and O
-
protecting groups in the glycosyl donor on yields of products and stereoselec-
tivity of glycosylation has been demonstrated.
The reason for this high sensitivity of the sialylation outcome to a protect-
ing group pattern of sialyl donor is currently unknown, although various hy-
[
22–29]
[25–29]
potheses have been advanced.
According to the supramer approach,
various protecting groups are the sites capable of intermolecular interactions,
which may determine the structure of supramer formed in each particular case
[
29]
and hence its reactivity.
For this reason, rational introduction of different
protecting groups at specific positions of the sialyl donor molecule may change
the structure of supramers formed and hence might be a novel way of modu-
lating its reactivity and stereoselectivity.
Such an approach requires reliable access to the O-unprotected sialic acid
derivatives with various N-protecting groups. One of the possible key building
blocks for the preparation of these derivatives could be a sialic acid tetrahy-
droxy amine like 2 (Sch. 1) or 5 (Sch. 2). In fact, both α- and β-anomers of
[
18,30]
[13,31]
[18]
amines 2
and 5
and related compounds
with other aglycons are
known and have been successfully used for the preparation of selectively pro-
tected sialyl donors with various protecting groups. Such compounds are com-
monly generated by acidic methanolysis of per-N,O-acetylated derivatives such
as 1[ (Sch. 1) and 4
32]
[33]
(Sch. 2) and used in situ for the introduction of the de-
sired N- and O-protecting groups. The target products have been isolated by a
combination of standard extractive workup and silica gel chromatography.
Scheme 1: The known[19,34] synthesis of neuraminic acid p-tolyl thioglycoside 3. Reagents
and conditions: a. (1) MsOH, MeOH, ꢀ; (2) treatment with anion-exchange resin; (3)
chromatography on silica gel (55%). b. CF CO Et, Et N, MeOH (94%). TFA = CF CO,
3
2
3
3
Tol = 4-methylphenyl.
For the synthesis of derivatives, which can be purified by chromatography
and survive aqueous workup, such an approach is quite reasonable. However,

N-Trifluoroacetylated Neuraminic Acid Tetraol
2
15
Scheme 2: Synthesis of neuraminic acid phenyl thioglycoside 6a, 6b and O-acyl derivatives
7
and 8. Reagents and conditions: a. (1) MsOH, MeOH, ꢀ; (2) extraction with Et O. b.
2
CF CO Et, Et N, CH Cl . c. Chromatography and crystallization (combined isolated yield of
3
2
3
2
2
◦
6
a and 6b is 71% from anomeric mixture 4a,b). d. CCl COCl, Py, CH Cl , 0→20 C, 14 h
3
2
2
◦
(
95%). e. (CF CO) O, CF CO Na (cat.), 0 C, 30 min (99%). f. (1) MsOH, MeOH, ꢀ; (2)
3 2 3 2
extraction with Et O; (3) CF CO Et, N,N-diisopropylethylamine, MeOH; (4) chromatography
2
3
2
and crystallization (89% from β-anomer 4b). TCA = CCl CO, TFA = CF CO.
3
3
for introduction of highly labile (acyl and probably silyl) groups, exclusion of
the possibility of hydrolysis and minimization of the number of operations dur-
ing isolation of the product are required. Therefore, it seems reasonable to have
at hand an N-protected sialic acid derivative with free hydroxyl groups that
can be purified by chromatography and/or crystallization prior to introduction
of O-protecting groups.
Indeed, a procedure for the synthesis of N-TFA tetraol 3 (Sch. 1) with p-
tolyl substituent at the sulfur atom and α-configuration of the glycoside bond
[
19]
was described recently.
The essential feature of this procedure was the use
[
34]
of pure amine precursor 2. After the deacetylation step, the reaction mixture
was treated with a large amount of an anion-exchange resin (in order to remove
10-fold excess of methanesulfonic acid), followed by column chromatography
on silica gel to give purified amine 2 in 55% yield. N-Trifluoroacetylation of
amine 2 gave tetraol 3 in 94% yield. The overall yield of tetraol 3 obtained by
this procedure was only 52%.
Here we describe an optimized practical synthesis of both α- and β-anomers
of related thioglycosides 6a and 6b (Sch. 2) on a multigram scale.

16 N. M. Podvalnyy et al.
2
RESULTS AND DISCUSSION
We started from the anomeric mixture of the known[33] peracetylated thiogly-
cosides 4a and 4b, which is difficult (although possible) to separate either by
crystallization or chromatography. We followed the commonly used procedure
[
13,18,30,31]
for acidic methanolysis of 4a,b and obtained (as others did
of amine 5a,b in the mixture with excess MsOH.
) the salt
The key feature of our approach is the modified procedure for removal of
excess MsOH, which is required for methanolysis of O- and N-acetyl groups.
As was mentioned above, at the step of acidic methanolysis of O- and N-acetyl
groups of 4a,b, treatment of the reaction mixture containing 10-fold excess of
MsOH with an anion-exchange resin and a column chromatography would lead
[
19,34]
to significant losses.
This problem was obviated by removal of the excess
MsOH by extraction with diethyl ether. Then the crude O- and N-deprotected
thiosialoside amine 5a,b in the form of the salt with MsOH was subjected to
N-trifluoroacetylation with ethyl trifluoroacetate in the presence of Et3N. At
this step it was found convenient to separate the anomers of N-trifluoroacetyl
derivative 6a and 6b by silica gel column chromatography and crystallization.
It is interesting to note that compound 6b crystallizes only after addition of
methanol to the solution, forming a solvate with methanol. This important
feature of 6b allows efficient crystallization of 6a from the anomeric mixtures.
The combined yield of 6a and 6b obtained from the anomeric mixture 4a,b
was 71% on a 16-g scale. When the individual β-isomer 4b was involved in our
procedure, separation of anomers was not required, which led to the increased
yield (89%) of the target tetraol 6b.
Elaboration of 6a and 6b in pure form allows access to a number of com-
pounds containing labile acyl groups. Synthesis of sialyl donors with labile
O-protecting groups like 7 and 8 from 5 or 6, prepared in situ using the pre-
viously developed methods, would be impossible due to their instability under
chromatographic purification on silica gel.
O-Trichloroacetate 7 was obtained by treatment of 6b with CCl3COCl in
dichloromethane in the presence of pyridine. Chromatographic purification of
7
on silica gel was possible but associated with losses due to hydrolysis. An
alternative workup procedure involving liquid–solid extraction with toluene
most of the salts remain in the solid residue), liquid–liquid extraction with
(
cold aqueous NaHCO3 and NaHSO4 solutions, and quick filtration through a
very short (3-mm) layer of silica gel enabled us to obtain the pure product 7 in
95% yield.
O-Trifluoroacetylation of 6b with (CF3CO)2O in the presence of soluble
[35]
bases (Py or Et3N)
did not allow isolation of the product 8 in pure form ap-
parently due to hydrolysis of O-trifluoroacetyl groups during aqueous extrac-
tions. Chromatography on silica gel was also absolutely unacceptable because
of the known lability of O-trifluoroacetyl groups.[ We managed to obtain pure
36]

N-Trifluoroacetylated Neuraminic Acid Tetraol
2
17
8
by conducting the acylation of 6b in the presence of CF3CO2Na as a base[37]
with subsequent removal of volatile components and extraction of the product
with warm anhydrous benzene. Five distinct signals of trifluoroacetyl groups
1
9
[38,39]
are present in the F NMR spectrum of compound 8. It was earlier noted
1
9
that F NMR spectra of trifluoroacetates of methyl glycosides can be used for
identification purposes.
CONCLUSIONS
In summary, a convenient and efficient method for the synthesis and
separation of α- and β-anomers of methyl (phenyl 3,5-dideoxy-2-thio-5-
trifluoroacetamido-D-glycero-D-galacto-nonulopyranosid)onate 6a and 6b on a
multigram scale was developed. It was shown that the obtained thiosialoside
tetraol can be utilized as a direct precursor of sialyl derivatives containing
labile O-acyl groups.
EXPERIMENTAL
General Methods
All reactions were carried out under argon atmosphere with anhydrous
solvents. The reactions were performed with the use of commercial reagents
(Aldrich, Fluka, Acros Organics) and distilled solvents purified and dried
according to standard procedures. Column chromatography was performed on
silica gel 60 (40–63 μm, Merck). Thin-layer chromatography was carried out
on plates with silica gel 60 F254 on glass or on aluminum foil (Merck). Spots
of compounds were visualized under UV light and by heating the plates after
1
immersion in a 1:10 (v/v) mixture of 85% aqueous H3PO4 and 95% EtOH. H,
1
3
19
C, and F NMR spectra were recorded for solutions in CDCl3 or CD3OD on
a Bruker AC-200 instrument (200.13, 50.32, and 188.31 MHz, respectively),
a Bruker AM-300 instrument (300.13, 75.48, and 282.38 MHz, respectively),
or a Bruker AVANCE 600 spectrometer (600.13, 150.9, and 564.69 MHz, re-
1
spectively). The H NMR chemical shifts are referred to the residual sig-
1
3
nal of CHCl3 (δH 7.27) or CHD2OD (δH 3.31) and the C NMR shifts to the
CDCl3 signal (δ 77.0) or CD3OD signal (δ 49.0). Assignments of the signals
C
C
in the NMR spectra were performed using 2D-spectroscopy (COSY, HSQC,
HMBC) and DEPT-135 experiments. High-resolution mass spectra (electro-
spray ionization, HR-ESI-MS) were measured in a positive mode on a Bruker
−5
micrOTOF II mass spectrometer for 2 × 10 M solutions in MeCN or MeOH.
Optical rotations were measured using a PU-07 automatic digital polarimeter
(
Russia).

18 N. M. Podvalnyy et al.
2
Methyl (phenyl 3,5-dideoxy-2-thio-5-trifluoroacetamido-α- and
β-D-glycero-D-galacto-nonulopyranosid)onate (6a and 6b)
Method A. To a stirred solution of the anomeric mixture of 4a and 4b
[
33]
(
(
16.0 g, 27.4 mmol, α:β ∼ 1:4.5)
in anhydrous MeOH (274 mL), neat MsOH
17.8 mL, 274 mmol) was added. The reaction mixture was heated under ar-
gon at 67 C (bath temperature) for 24 h with a reflux condenser, then cooled
to rt and concentrated under reduced pressure (bath temperature 40 C). The
◦
◦
syrupy residue was triturated with Et2O (100 mL) for 15 min followed by de-
cantation of the solvent, and the procedure was repeated three more times with
Et2O (3 × 100 mL) until most of the MsOH was extracted. The solid residue
(crude tetraol 2 containing free amino group in the form of the salt with MsOH)
was dried in vacuo and dissolved in MeOH (55 mL). Triethylamine (30.5 mL,
2
20 mmol) and CF3COOEt (17.1 mL, 144 mmol) were added in that order with
◦
stirring. After 24 h at rt (∼20 C), the reaction mixture was concentrated un-
der reduced pressure, and the residue was co-concentrated with MeCN (3 ×
20 mL) and dried in vacuo to give a crude anomeric mixture 6a,b as a white
solid (33.7 g).
At the first step of isolation, the anomeric mixture 6a,b was purified by
chromatography on silica gel with gradient elution (acetone–toluene 3:7 v/v
100% acetone). Fractions containing β-anomer 6b as the major component
→
(
R = 0.45, CHCl3–MeOH, 4:1 v/v) were combined and concentrated under re-
f
duced pressure; the residue was dissolved in acetone (25 mL) and diluted with
Et2O (50 mL); and then petroleum ether–Et2O mixture (1:1 v/v) was added
dropwise until a slight turbidity appeared. After addition of MeOH (5 mL),
colorless crystals (prisms) started to precipitate. After precipitation of a ma-
jor part of the product (∼1 h), an additional amount of petroleum ether–Et2O
(1:1 v/v, total 100 mL) was added dropwise to the mother liquor containing
◦
crystals, and the mixture was kept at +4 C overnight. The product was fil-
tered off and air-dried to give β-anomer 6b as a solvate with MeOH contain-
ing a minor admixture of α-anomer. The crystalline product was dissolved
in a minimal volume of acetone, the solution was concentrated to dryness,
and the residue was recrystallized one more time as mentioned above, lead-
ing to pure β-anomer 6b as a 1:1 solvate with MeOH (6b·MeOH) (6.70 g,
49%).
At the second step of isolation, the remaining chromatographic fractions
were combined with all mother liquors from the previous crystallizations,
concentrated under reduced pressure, and dried in vacuo. The residue was
purified by column chromatography on silica gel with gradient elution with
CHCl3–acetone (3:1→1:1 v/v). Fractions containing α-anomer 6a (Rf = 0.38,
CHCl3–acetone 1:1 v/v) as the major component were combined, concentrated
under reduced pressure, and dried in vacuo. The residue was dissolved in
acetone (10 mL) and diluted with Et2O (20 mL), and then the petroleum

N-Trifluoroacetylated Neuraminic Acid Tetraol
2
19
ether–Et2O mixture (1:1 v/v) was added dropwise until a slight turbidity ap-
peared. Colorless crystals formed were collected and dried in vacuo to give pure
α-anomer 6a (1.72 g, 13%).
The remaining fractions of the second chromatography and the mother
liquor from the previous crystallization were combined, concentrated under
reduced pressure, and dried in vacuo. The residue was dissolved in acetone
(
10 mL) and diluted with Et2O (20 mL), and then the petroleum ether–Et2O
mixture (1:1 v/v) was added until a slight turbidity appeared. After addition of
MeOH (2 mL), the product (6b·MeOH) precipitated. Colorless crystals were
collected and air-dried, leading to an additional amount of pure β-anomer
6
b as solvate with MeOH (6b·MeOH) (1.26 g, 58% [overall yield of β-anomer
6b]).
◦
Data for 6a: Rf 0.38 (CHCl3–acetone, 1:1). m.p. 191–195 C (from
2
2
acetone–Et2O–petroleum ether). [α]D +55.0 (c 1.0, MeOH). HR-ESI-MS:
+
found m/z 492.0913 [M + Na] . Calcd for C18H22F3NO8SNa: 492.0910. Ele-
mental analysis: calcd (%) for C18H22F3NO8S (469.43): C 46.05, H 4.72, N 2.98;
1
found: C 46.08, H 4.86, N 2.99. H NMR (600 MHz, CD3OD): δ ppm 7.56–7.59
(
m, 2 H, Ph), 7.41–7.45 (m, 1 H, Ph), 7.35–7.39 (m, 2 H, Ph), 4.02 (dd∼t, J4,5
10.3 Hz, J5,6 = 10.3 Hz, 1 H, H-5), 3.76–3.81 (m, 2 H, H-8, H-9b), 3.69 (ddd,
J3ax,4 = 11.6 Hz, J4,5 = 10.3 Hz, J3eq,4 = 4.8 Hz, 1 H, H-4), 3.67 (dd, J5,6 =
0.3 Hz, J6,7 = 1.6 Hz, 1 H, H-6), 3.62 (s, 3 H, OCH3), 3.59–3.62 (m, 1 H,
=
1
H-9a), 3.44 (dd, J7,8 = 9.0 Hz, J6,7 = 1.6 Hz, 1 H, H-7), 2.85 (dd, J3ax,3eq =
1
1
2.8 Hz, J3eq,4 = 4.8 Hz, 1 H, H-3eq), 1.90 (dd, J3ax,3eq = 12.8 Hz, J3ax,4 =
1.6 Hz, 1 H, H-3ax). 19F NMR (282 MHz, CD3OD) δ ppm −77.07. C NMR
13
3
(151 MHz, CD3OD): δ 171.0 (COOCH3), 159.8 (q, J
= 36.8 Hz, COCF3),
C,F
2
1
38.0, 131.3, 130.3, 130.1 (Ph), 117.6 (q, JC,F = 287.2 Hz, CF3), 88.2 (C-2), 76.4
(C-6), 73.4 (C-8), 70.2 (C-7), 69.1 (C-4), 64.6 (C-9), 53.8 (C-5), 53.4 (OCH3), 41.9
C-3).
(
Data for 6b·MeOH: R = 0.19, CHCl3–acetone (1:1 v/v). R 0.45,
f
f
◦
CHCl3–MeOH (4:1 v/v). m.p. 114–118 C (crystal solvate with MeOH from
2
2
acetone–Et2O–petroleum ether–MeOH). [α]D −141.2 (c 1.0, MeOH). HR-ESI-
+
MS: found m/z 492.0905 [M + Na – MeOH] . Calcd for C18H22F3NO8SNa:
4
92.0910. Elemental analysis: calcd (%) for C19H26F3NO9S (501.47): C 45.51,
1
H 5.23, N 2.79; found: C 45.44, H 5.02, N 2.83. H NMR (600 MHz, CD3OD): δ
ppm 7.58–7.61 (m, 2 H, Ph), 7.33–7.40 (m, 3 H, Ph), 4.72 (dd, J5,6 = 10.6 Hz,
J6,7 = 0.8 Hz, 1 H, H-6), 4.20 (ddd, J3ax,4 = 11.7 Hz, J4,5 = 10.1 Hz, J3eq,4 =
4
.7 Hz, 1 H, H-4), 4.06 (dd∼t, J5,6 = 10.6 Hz, J4,5 = 10.1 Hz, 1 H, H-5), 3.82
(dd, J
= 11.4 Hz, J8,9a = 2.9 Hz, 1 H, H-9a), 3.77 (ddd, J7,8 = 9.2 Hz, J
9
a,9b
8,9b
=
5.5 Hz, J8,9a = 2.9 Hz, 1 H, H-8), 3.67 (dd, J
= 11.4 Hz, J
= 5.5 Hz,
9
a,9b
8,9b
1
H, H-9b), 3.53 (s, 3 H, OCH3), 3.50 (d, J7,8 = 9.2 Hz, 1 H, H-7), 2.70 (dd,
J3ax,3eq = 13.6 Hz, J3eq,4 = 4.7 Hz, 1 H, H-3eq), 1.99 (dd, J3ax,3eq = 13.6 Hz,
1
9
13
J3ax,4 = 11.7 Hz, 1 H, H-3ax). F NMR (282 MHz, CD3OD): δ ppm −76.89.
C
3
NMR (151 MHz, CD3OD): δ ppm 171.0 (COOCH3), 159.9 (q, J
= 36.9 Hz,
C,F

20 N. M. Podvalnyy et al.
2
OCOCF3), 137.4, 131.5, 130.7, 130.1 (Ph), 117.7 (q, 2JC,F = 287.3 Hz, CF3), 91.6
(
(
C-2), 72.8 (C-6), 71.6 (C-8), 70.9 (C-7), 67.8 (C-4), 65.2 (C-9), 54.6 (C-5), 53.2
OCH3), 42.4 (C-3).
Method B. To a stirred solution of 4b (300 mg, 0.514 mmol)[33] in anhy-
drous MeOH (12 mL), neat MsOH (0.34 mL, 5.2 mmol) was added. The reac-
◦
tion mixture was heated under argon at 67 C (bath temperature) with a re-
flux condenser for 24 h, cooled to rt, and concentrated under reduced pressure
◦
(
bath temperature 40 C). The syrupy residue was triturated with Et2O (10 mL)
for 15 min followed by decantation of the solvent, and the procedure was re-
peated three more times with Et2O (3 × 5 mL) until most of the MsOH was
extracted. The solid residue was dried in vacuo and dissolved in MeOH (4 mL).
N,N-Diisopropylethylamine (0.72 mL, 4.1 mmol) and CF3COOEt (0.32 mL,
◦
2
.7 mmol) were added in that order with stirring. After 24 h at rt (∼20 C), the
reaction mixture was concentrated under reduced pressure, and the residue
was co-concentrated with MeCN (3 × 3 mL) and dried in vacuo to give crude
6b as a white solid.
The product was purified by column chromatography on silica gel with gra-
dient elution (acetone–toluene 1:3 v/v → 100% acetone). Fractions containing
the product (R 0.45, CHCl3–MeOH, 4:1) were combined and concentrated un-
f
der reduced pressure; the residue was dissolved in acetone (1 mL) and diluted
with CH2Cl2 (2 mL) and Et2O (3 mL); then petroleum ether was added drop-
wise until a slight turbidity appeared. After addition of MeOH (0.2 mL), col-
orless prisms started to precipitate. After precipitation of a major part of the
product, an additional amount of petroleum ether (total 8 mL) was added drop-
◦
wise and the mixture was kept at +4 C overnight. The crystals were filtered
off and air-dried to afford the target product 6b in the form of solvate with
MeOH (6b·MeOH) (230 mg, 89%).
Methyl (phenyl 3,5-dideoxy-2-thio-4,7,8,9-tetra-O-
trichloroacetyl-5-trifluoroacetamido-D-glycero-β-
D-galacto-nonulopyranosid)onate (7)
Tetraol 6b·MeOH (121.0 mg, 0.241 mmol) was co-concentrated with ace-
tone (2 × 5 mL) and dried in vacuo for ∼1 h; then anhydrous CH2Cl2 (5 mL)
was added followed by pyridine (240 μL, 2.97 mmol); and the resulting sus-
◦
pension was cooled to 0 C (ice–water bath). Trichloroacetyl chloride (160 μL,
1.425 mmol) was added dropwise under argon (after addition the starting ma-
terial has dissolved); the reaction mixture was allowed to warm to rt and
◦
stirred for 16 h at ∼20 C, then concentrated under reduced pressure, co-
concentrated with toluene (2 × 10 mL), and dried in vacuo. The residue was
triturated with toluene (5 mL), and the solids were filtered off and washed
with toluene (2 × 5 mL). The combined filtrate was concentrated to dryness,
and the residue was dissolved in toluene (5 mL), washed with ice-cold water

N-Trifluoroacetylated Neuraminic Acid Tetraol
2
21
(5 mL), ice-cold saturated aqueous NaHCO3 (5 mL), and ice-cold 1 M aque-
ous NaHSO4 (5 mL). Back-extraction with ∼1.5 mL of toluene at each step
was performed. The combined organic layer was washed with ice-cold water
(
5 mL), dried over Na2SO4, and quickly filtered through a very short (∼3 mm)
layer of silica gel; solids were washed with the 19:1 toluene–EtOAc mixture
10 mL) and concentrated under reduced pressure, and the residue was dried
in vacuo to give 7 as a white foam (242.6 mg, 95%).
(
1
8
[
α]D −66.9 (c 1.0, CHCl3). HR-ESI-MS: found m/z 1062.7108 [M +
+
1
NH4] . Calcd for C26H22Cl12F3N2O12S: 1062.7102. H NMR (300 MHz, CDCl3):
δ ppm 7.38–7.60 (m, 5 H, Ph), 6.66 (d, J5,NH = 9.6 Hz, 1 H, NH), 5.80 (ddd∼td,
J3ax,4 = 11.6 Hz, J4,5 = 10.9 Hz, J3eq,4 = 4.9 Hz, 1 H, H-4), 5.63 (dd∼t, J6,7 =
2
.2 Hz, J7,8 = 2.2 Hz, 1 H, H-7), 5.13 (ddd∼dt, J
= 9.6 Hz, J8,9a = 2.4 Hz,
8
,9b
J7,8 = 2.1 Hz, 1 H, H-8), 5.06 (dd, J5,6 = 10.5 Hz, J6,7 = 2.2 Hz, 1 H, H-6), 4.82
dd, J = 12.4 Hz,
= 12.4 Hz, J8,9a = 2.4 Hz, 1 H, H-9a), 4.59 (dd, J
(
9
a,9b
9a,9b
J8,9b = 9.6 Hz, 1 H, H-9b), 4.23 (ddd∼q, J4,5 = 10.9 Hz, J5,6 = 10.5 Hz, J5,NH
=
9.6 Hz, 1 H, H-5), 3.77 (s, 3 H, OCH3), 2.97 (dd, J3ax,3eq = 13.9 Hz, J3eq,4 =
4
.9 Hz, 1 H, H-3eq), 2.35 (dd, J3ax,3eq = 13.9 Hz, J3ax,4 = 11.6 Hz, 1 H, H-3ax).
1
9
13
F NMR (282 MHz, CDCl3): δ ppm −75.71. C NMR (75 MHz, CDCl3): δ ppm
3
1
1
7
3
67.4, 162.1, 161.4, 160.8, 160.8 (CO), 158.5 (q, J
= 38.7 Hz, OCOCF3),
C,F
2
36.0, 131.0, 129.8, 127.2 (Ph), 115.1 (q, JC,F = 285.0 Hz, CF3), 88.0 (C-2),
6.5 (C-6), 73.1, 73.0 (C-4, C-7), 71.3 (C-8), 65.2 (C-9), 53.2 (OCH3), 50.2 (C-5),
6.8 (C-3).
Methyl (phenyl 3,5-dideoxy-2-thio-4,7,8,9-tetra-O-
trifluoroacetyl-5-trifluoroacetamido-D-glycero-β-D-
galacto-nonulopyranosid)onate (8)
Tetraol 6b·MeOH (240.0 mg, 0.47 mmol) was co-concentrated with anhy-
drous MeCN (2 × 10 mL) and dried in vacuo. Sodium trifluoroacetate (80 mg,
◦
0
.58 mmol, dried at 80 C in vacuo for 1 h) was added, and the reaction flask
◦
was flushed with argon and cooled to 0 C. Trifluoroacetic anhydride (6.4 mL)
was added, and the reaction mixture was stirred for 30 min at 0 C (ice–water
◦
bath) and then concentrated to dryness; the residue was triturated with warm
◦
(
+40 C) anhydrous benzene (2 × 10 mL), the extracts were filtered through
a silanized glass wool (Serva) plug, solids were washed with warm anhydrous
benzene, and the combined filtrate was concentrated and dried in vacuo to give
per-trifluoroacetylated compound 8 as a white foam (404.4 mg, 99%). (Cau-
tion! Benzene has been identified as a carcinogen. All procedures involving
benzene should be carried out in a well-ventilated hood, and glove protection
is required.) The product shows limited solubility in CDCl3 (after addition of
CDCl3 to the sample of 8 it starts to crystallize).
◦
20
m.p. 191–192 C (from CDCl3). [α]D −102.1 (c 2.04, CHCl3). HR-ESI-MS:
+
1
found m/z 891.9946 [M + K] . Calcd for C26H18F15NO12SK: 891.9942. H NMR

22 N. M. Podvalnyy et al.
2
(
300 MHz, CDCl3): δ ppm 7.37–7.50 (m, 5 H, Ph), 6.55 (d, J5,NH = 9.9 Hz, 1 H,
NH), 5.69–5.80 (m, 1 H, H-4), 5.55 (dd∼t, J7,8 = 1.8 Hz, J6,7 = 2.2 Hz, 1 H,
H-7), 4.92 (dd, J5,6 = 10.5 Hz, J6,7 = 2.2 Hz, 1 H, H-6), 4.98–5.04 (m, 1 H, H-8),
4
.72 (dd, J9
= 12.5 Hz, J
= 2.6 Hz, 1 H, H-9b), 4.29 (dd, J
= 12.5 Hz,
a,9b
8,9b
9a,9b
J8,9a = 9.1 Hz, 1 H, H-9a), 4.18 (ddd∼q, J5,6 = 10.5 Hz, J4,5 = 10.2 Hz, J5,NH
=
9.9 Hz, 1 H, H-5), 3.80 (s, 3 H, OCH3), 2.92 (dd, J3ax,3eq = 14.1 Hz, J3eq,4 =
5
.0 Hz, 1 H, H-3eq), 2.36 (dd, J3ax,3eq = 14.1 Hz, J3ax,4 = 11.6 Hz, 1 H, H-3ax).
1
1
9
F NMR (282 MHz, CDCl3): δ ppm −74.13, −74.68, −74.71, −74.84, −75.75.
3
C NMR (75 MHz, CDCl3): δ 167.4 (CO), 158.2–155.5 (m, 5 × COCF3), 136.0,
31.0, 129.7, 127.0 (Ph), 119.7–110.6 (m, 5 × COCF3), 87.5 (C-2), 75.0 (C-6),
1.9, 71.5, 71.2 (C-4, C-7, C-8), 63.8 (C-9), 53.3 (OCH3), 49.8 (C-5), 36.5 (C-3).
1
7
ACKNOWLEDGMENTS
The authors would like to acknowledge Dr. A. O. Chizhov and Dr. V. I. Bra-
gin for the recording of high-resolution mass spectra and 2D NMR spectra,
respectively.
FUNDING
This work was supported by the Russian Scientific Foundation (Project No.
14-13-00978).
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