Differences in reactivity of N-acetyl- and N,N-diacetylsialyl chlorides caused by their different supramolecular organization in solutions

Article abstract of DOI:10.1007/s11172-017-1999-x

O-Sialylation of a substituted indolin-3-one under phase-transfer catalysis conditions, which does not occur when N-acetylsialyl chloride is used, proceeds with N,N-diacetylsialyl chloride as the glycosyl donor. A study using dynamic light scattering of solutions of both sialyl chlorides under conditions close to the conditions used for glycosylation showed a difference in the correlation radii of light scattering particles in such solutions. This suggests that the introduction of an additional N-acetyl group into the sialyl chloride significantly alters the structure of the supramers of glycosyl donor, which apparently have an increased accessibility of individual molecules for the attack by a nucleophile, which increases its reactivity.

Full text of DOI:10.1007/s11172-017-1999-x

Russian Chemical Bulletin, International Edition, Vol. 66, No.11, pp. 2173—2179, November, 2017
2173
Differences in reactivity of Nꢀacetylꢀ and N,Nꢀdiacetylsialyl chlorides
caused by their different supramolecular organization in solutions
A. V. Orlova,a T. V. Laptinskaya,b N. V. Bovin,c and L. O. Kononovа
аN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Eꢀmail: kononov@ioc.ac.ru, leonid.kononov@gmail.com
bM. V. Lomonosov Moscow State University, Department of Physics,
Build. 2, 1 Leninskie Gory, 119991 Moscow, Russian Federation
cM. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences,
16/10 ul. MiklukhoꢀMaklaya, 117997 Moscow, Russian Federation
OꢀSialylation of a substituted indolinꢀ3ꢀone under phaseꢀtransfer catalysis conditions,
which does not occur when Nꢀacetylsialyl chloride is used, proceeds with N,Nꢀdiacetylsialyl
chloride as the glycosyl donor. A study using dynamic light scattering of solutions of both
sialyl chlorides under conditions close to the conditions used for glycosylation showed
a difference in the correlation radii of light scattering particles in such solutions. This suggests
that the introduction of an additional Nꢀacetyl group into the sialyl chloride significantly
alters the structure of the supramers of glycosyl donor, which apparently have an increased
accessibility of individual molecules for the attack by a nucleophile, which increases its
reactivity.
Key words: N,Nꢀdiacetylsialyl chloride, sialylation, dynamic light scattering, chromoꢀ
genic substrates, phaseꢀtransfer catalysis, mesoscale particles, supramers, solution structure,
reactivity.
Complex carbohydrate molecules containing sialic
acid moieties (including Nꢀacetylneuraminic acid) are
important natural compounds determining the course
of a number of immunological, neurobiological, oncoꢀ
logical and other biological processes.1—8 Synthesis of
glycoconjugates, oligosaccharides and their analogs conꢀ
taining sialic acids, as well as the study of enzymes cleavꢀ
ing the glycosidic bonds formed by sialic acids (sialidasꢀ
es, neuraminidases, neuraminosyl glycohydrolase, EC
3.2.1.18) is the most important and sometimes the only
instrument for the research in the listed above areas of
fundamental glycobiology (see recent reviews 9—11).
It is convenient to detect neuraminidases of viruses,
bacteria, and mammals12—15 using chromogenic subꢀ
R = H, Na
strates,12,16—19 such as (indolꢀ3ꢀyl)sialosides 112,16,18
and 2,19—21 which form colored products after cleavage
with enzymes.13,19
tecting groups.21 All known methods for the preparaꢀ
There are several methods for the synthesis of sialoꢀ
sides 1 and 2 and their analogs: glycosylation of 3ꢀacetꢀ
oxyꢀ1ꢀacetylꢀ5ꢀbromoindole (3)12,16,17 and 3ꢀacetoxyꢀ
1ꢀacetylꢀ5ꢀbromoꢀ4ꢀchloroindole (4)20 in an alkaline
medium and glycosylation of allyl 5ꢀbromoꢀ4ꢀchloroꢀ3ꢀ
hydroxyindoleꢀ2ꢀcarboxylate (5) under the phaseꢀtransꢀ
fer catalysis conditions with subsequent removal of proꢀ
tion of these chromogenic substrates are based on the use
of Nꢀacetylsialyl chloride 622—27 with one acetyl group at
the N(5) atom as a glycosyl donor. Recently, we suggestꢀ
ed28 a new sialyl donor, namely, N,Nꢀdiacetylsialyl chloꢀ
ride 7, and showed its advantages in comparison with
traditional Nꢀacetylsialyl chloride 6, especially for the
glycosylation of negatively charged glycosyl acceptors,
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2173—2179, November, 2017.
1066ꢀ5285/17/6611ꢀ2173 © 2017 Springer Science+Business Media, Inc.

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Orlova et al.
including glycosylation under phaseꢀtransfer catalyꢀ
sis conditions.
with N,Nꢀdiacetylsialyl chloride 7 and Nꢀacetylsialyl
chloride 6.
Results and Discussion
Glycosylation of indolinꢀ3ꢀone 8 with sialyl chlorides
6 and 7 was carried out in a twoꢀphase system (EtOAc—
1 М aqueous solution of Na2CO3) in the presence of
a phaseꢀtransfer catalyst (Bu4NHSO4). Selective detecꢀ
tion on TLC (details are given in the Experimental secꢀ
tion) showed that the compounds simultaneously conꢀ
taining an indole fragment and a monosaccharide residue
are absent in the reaction mixture of glycosylation of inꢀ
dolinꢀ3ꢀone 8 with Nꢀacetylsialyl chloride 6. This indiꢀ
cates that the reaction of Nꢀacetylsialyl chloride 6 with
Nꢀacetylindoxyl derivative 8 does not occur. At the same
time, glycosylation of derivative 8 with N,Nꢀdiacetylsialyl
chloride 7 leads to a mixture of the target glycoside 9 and
its partially Nꢀdeacetylated glycosides 10 and 11, as
well as to the corresponding glycals 1230 and 1327,31,32
(Scheme 1), which were isolated* by chromatography on
silica gel in low yields.
X = H (3), Cl (4); R = H (6), Ac (7)
In the present work, we planned to find out possible
differences in the results of glycosylation of 1ꢀacetylꢀ
5ꢀbromoꢀ4ꢀchloroindolinꢀ3ꢀone (8),29 which is a ketoꢀ
tautomer of the corresponding 3ꢀhydroxy derivative,
Scheme 1
R = H (2a), NBu4 (2b);
R´ = R´´ = Ac (9), R´ = Ac, R´´ = H (10), R´ = H, R´´ = Ac (11);
R´´´ = Ac (12), H (13)
Reagents and conditions: a. 1) 80% H2SO4; 2) H2O; b. 1) Bu4NHSO4, EtOAc—1 M Na2CO3; 2) extraction with EtOAc. c. Et3N—
MeOH—H2O.
* Indolyl sialosides 9 and 11 were obtained as the mixtures with N,Nꢀdiacetylglycal 12 and Nꢀacetylglycal 13, respectively.

Reactivity and solution structure
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 11, November, 2017 2175
The formation of derivatives with different degree of
Nꢀacetylation under basic reaction conditions suggests
that it is reasonable to avoid chromatographic isolation
at this stage in order to reduce losses, especially given
that these Nꢀacetyl groups are destinied to be removed in
further steps. Therefore, after the glycosylation reaction
of Nꢀacetylindoxyl derivative 8 with N,Nꢀdiacetylsialyl
chloride 7, removal of salts (extraction with ethyl aceꢀ
tate) and solvents, the residue without additional purifiꢀ
cation was subjected to the reaction of removal of all
Оꢀ and Nꢀprotecting groups (Et3N—MeOH—H2O).*
Subsequent concentration of the reaction mixture and
chromatography on silica gel afforded the target sialoside 2
in 38% yield (see Scheme 1).
The absence of glycosylation reaction of Nꢀacetylinꢀ
doxyl derivative 8 with Nꢀacetylsialyl chloride 6 (in conꢀ
trast to the reaction with N,Nꢀdiacetylsialyl chloride 7)
requires a special discussion. An increased reactivity of
sialyl donors with two acetyl groups at the N(5) nitrogen
atom is known.33,34 Recently, it was shown35,36 that the
presence of the N,Nꢀdiacetylneuraminic acid moiety even
in the disaccharide glycosyl donor significantly increases
both its reactivity and yields of glycosylation products as
compared to the results of the reactions involving otherꢀ
wise protected derivatives of related structure, though the
N,Nꢀdiacetylamino group was located on the nonꢀreducꢀ
ing end of the disaccharide and, therefore, significantly
remote from the reaction center.
Recently, we hypothesized that intermolecular interꢀ
actions of reagents in solutions can lead to supramolecuꢀ
lar aggregation in the reaction mixture, which, in turn,
causes the formation of solutions with a modified strucꢀ
ture (see review37). According to this "supramer" apꢀ
proach,37—50 we believe that in this reaction, as in the
case of other sialyl donors,28,37,39,41—44,50 such signifiꢀ
cant differences in the reactivity of sialyl chlorides 6 and
7 with one and two acetyl groups at the N(5) nitrogen
atom are associated with differences in the structure of
the corresponding reaction solutions and, in particular,
with the structural features of the glycosyl donor supraꢀ
mers.37 In fact, the intermolecular hydrogen bonds involvꢀ
ing sialyl donor molecules39,41,50 are possible only in soluꢀ
tions of Nꢀacetylsialyl chloride 6, the molecules of which
contain an NH group, resulting in the decrease in its
reactivity39,41,44 due to the formation of hydrogenꢀbonded
supramers of the sialyl donor, in which the access to the
reaction center may be hindered37,44 (cf. Refs 35 and 36).
Moreover, recent studies51—55 showed that many
macroscopically homogeneous solutions used in routine
laboratory practice are not homogeneous, as it is comꢀ
monly assumed.37 In solution, depending on concentraꢀ
tion, solvent nature, temperature, and the presence of
other compounds, including impurities, a number of suꢀ
pramolecular species (supramers in our terminology37)
may exist, differing in structure and size and having chemꢀ
ical properties significantly different from those of the
parent molecules comprising the supramers.37 Recently,
it was shown57 that dynamic light scattering (DLS) is
a convenient tool for the analysis of reactivity features in
such systems56,57
.
Therefore, we compared the solutions of Nꢀacetylꢀ
and N,Nꢀdiacetylsialyl chlorides 6 and 7 using DLS unꢀ
der conditions close to those used for carrying out the
reactions with Nꢀacetylindoxyl derivative 8. As a first apꢀ
proximation, we investigated the solutions of sialyl chlorꢀ
ides in anhydrous ethyl acetate (Fig. 1). In the DLS exꢀ
periment, the correlation function for anhydrous ethyl
acetate indicated the absence of light scattering particles in
this solvent. At the same time, the correlation functions
for solutions of both sialyl chlorides 6 and 7 in anhydrous
ethyl acetate (in both cases the concentration was the
same, 86 mmol L–1) indicated the presence of light scatꢀ
g2(τ) – 1
0.3
a
1
2
3
0.2
0.1
10–3 10–2 10–1 100 101 102 103 104 τ/ms
I (arb. units)
1.0
b
1
2
3
0.8
0.6
0.4
0.2
10–1 100
101
102
103
104
105 R/nm
Fig. 1. The results of dynamic light scattering experiments for
anhydrous ethyl acetate (1), 86 mM solution of sialyl chloride 6
in anhydrous ethyl acetate (2), and 86 mM a solution of sialyl
chloride 7 in anhydrous ethyl acetate (3): correlation functions
(a) and size distribution of correlation radii (R ) of light scatterꢀ
ing particles (b).
* Under these conditions, the single acetyl group present in
various derivatives of Nꢀacetylneuraminic acid is stable.

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Orlova et al.
g2(τ) – 1
0.7
tering particles. It is important that these correlation funcꢀ
tions differed noticeably in their appearance (see Fig. 1,
a), apparently reflecting the differences in the structure
of the solutions of sialyl chlorides 6 and 7 in anhydrous
ethyl acetate. It is interesting that mesoscale particles of
similar size predominate in both cases: the correlation
radius is ~102 nm (see Fig. 1, b), as it was noted in previꢀ
ous studies.51—55,57
a
0.6
0.5
0.4
0.3
0.2
0.1
1
2
3
However, since sialylation reactions were carried out
under phaseꢀtransfer catalysis conditions in the twoꢀphase
system ethyl acetate—water, a more realistic model of the
reaction medium is ethyl acetate saturated with water,*
in which large particles with correlation radii of about
1 μm predominate according to the DLS data (Fig. 2).
Most likely, they correspond to microdroplets of water
dispersed in ethyl acetate (since before the measurements
all the samples were filtered through a filter with pores of
0.45 μm, these micrometer objects were formed already
after filtration; cf. Refs 58 and 59). Therefore, it is not
surprising that micrometer species also predominated in
solutions of sialyl chlorides 6 and 7 in ethyl acetate satuꢀ
rated with water (see Fig. 2). In this case, not only did the
correlation functions for these solutions differ noticeably
in appearance (see Fig. 2, a), but also the radii of the
lightꢀscattering particles in these solutions differed conꢀ
siderably in their value (see Fig. 2, b). In this case, the
change in the size of the observed species relative to the
size of the lightꢀscattering particles in the initial ethyl
acetate (in the absence of glycosyl chlorides) saturated
with water is different: a decrease in the particle size is
observed for the solution of Nꢀacetylsialyl chloride 6,
while the particle size increases for the solution of N,Nꢀdiꢀ
acetylsialyl chloride 7. This indicates that the sialyl
chloride molecules are components of these lightꢀscatꢀ
tering particles. We believe that the observed differences
are due to the fact that the molecules of N,Nꢀdiacetylsiaꢀ
lyl chloride 7 can act only as acceptors of hydrogen bonds,
while water molecules act as donors. At the same time,
the molecules of Nꢀacetylsialyl chloride 6, in contrast to
the molecules of N,Nꢀdiacetylsialyl chloride 7, can also
act as hydrogen bond donors and form hydrogen bonds
not only with solvent molecules, but also with each other.
It is interesting that smaller particles corresponding
to the denser** supramers of glycosyl donor are present in
the solution of Nꢀacetylsialyl chloride 6. Therefore, it is
not surprising that the reactivity of this substance is lower
than the reactivity of N,Nꢀdiacetylsialyl chloride 7, formꢀ
ing looser** suprameres of larger size, in which the indiꢀ
10–3 10–2 10–1 100 101 102 103
I ( arb. units )
104 τ/ms
b
1.0
0.8
0.6
0.4
0.2
1
2
3
100
101
102
103
R/nm
Fig. 2. The results of dynamic light scattering experiments for
anhydrous ethyl acetate saturated with water (1), 86 mM solution
of sialyl chloride 6 in ethyl acetate saturated with water (2), and
86 mM solution of sialyl chloride 7 in ethyl acetate saturated
with water (3): correlation functions (a) and size distribution of
correlation radii (R ) of light scattering particles (b).
vidual sialyl donor molecules are more accessible for the
attack by a nucleophile.
In conclusion, we observed a significant difference in
the reactivity of Nꢀacetylsialyl chloride 6 and N,Nꢀdiaceꢀ
tylsialyl chloride 7 in the Oꢀsialylation reaction of indoꢀ
linꢀ3ꢀone 8. A significantly greater reactivity of N,Nꢀdiꢀ
acetylsialyl chloride 7 can be associated with the different
structure of the reaction solutions of sialyl chlorides 6
and 7, namely, with different size and structure of the
supramers of sialyl chlorides 6 and 7, which was estabꢀ
lished by dynamic light scattering. The use of sialyl
chloride 7 with two Nꢀacetyl groups in these reactions
made it possible to obtain a chromogenic substrate of
neuraminidase 2, which is indeed hydrolyzed by the inꢀ
fluenza virus (details of these experiments will be pubꢀ
lished separately).
* Note that after filtration through a filter with pores of 0.45 μm,
the sample of ethyl acetate saturated with water was a macroꢀ
scopically homogeneous colorless and clear liquid.
** Our rough estimate of the relative density of supramers is
based on the assumption that a denser packing of equal number
of glycosyl donor molecules (note that the molar concentraꢀ
tions of the solutions under study are the same) should lead to
supramers of smaller size.
Experimental
The reactions were carried out using commercial reagents.
Indolinꢀ3ꢀone 8 was obtained according to the described proꢀ

Russian Chemical Bulletin, International Edition, Vol. 66, No.11, pp. 2177—2179, November, 2017
2177
cedure.29 Solvents were purified and distilled before use acꢀ
cording to the standard procedures. Anhydrous ethyl acetate
for carrying out glycosylation was obtained by distillation over
phosphorus pentoxide and stored over molecular sieves 4 Å.
Ethyl acetate saturated with water was obtained by addition of
residue in МеОН (3 mL). After 1 h, the mixture was diluted
with H2O (5.1 mL). After 16 h, the solvents were removed on a
rotary evaporator. The residue was subjected to chromatograꢀ
phy (gradient: PriOH—EtOAc—H2O (2 : 6 : 1) → PriOH—
EtOAc—H2O (2 : 3 : 1)) to isolate the target product 2 (87.8 mg,
38%) as a mixture of* an acid and a tetrabutylammonium salt in
the ratio of 1 : 7 (1H NMR data). Rf 0.50 (PriOH—EtOAc—
freshly prepared ultrapure water (5 mL; resistivity >18 MΩ cm–1
)
to anhydrous ethyl acetate (5 mL) and the resulting mixture
was vigorously shaken for 2 min. Once the layers were formed
(10 min later), the upper (organic) phase was separated and
filtered (4 times) through a membrane filter (0.45 μm, PTFE,
Supelco) to obtain a macroscopically uniform colorless and
clear liquid. TLC is performed on DCꢀAlufolien Kieselgel 60
F254 plates (Merck) in the solvent systems indicated in the deꢀ
scription of the experiment for each individual case. Chroꢀ
matographic zones of carbohydrateꢀcontaining substances were
visualizes by the immersion of the plate into a solution of 85%
H3PO4 in 96% EtOH (1 : 10, v/v), followed by its heating to
150 °C. Under these conditions, the zones of substances conꢀ
taining an indoxyl fragment acquired a deep blue color, which
in the case of carbohydrate derivatives turned black ("charred")
upon further heating. The zones of UV absorbing compounds
were detected by absorption at 254 nm. Preparative chromatoꢀ
graphic separation was carried out on columns with silica gel 60
H2O, 2 : 2 : 1). 1H NMR (CD3OD), δ: 1.01 (t, 12 H, MeBu N
,
J = 8.0 Hz); 1.29—1.50 (m, 8 H, CH2MeBu N); 1.54—14.73
4
(m, 8 H, CH2CH2MeBu N); 1.98—2.03 (m, 4 H, CH3CON,
Hax(3)); 3.00 (dd, 1 H, 4Heq(3), J = 12.0 Hz, J = 4.6 Hz);
3.14—3.27 (m, 8 H, CH2N); 3.50—3.98 (m, 7 H, H(4), H(5),
H(6), H(7), H(8), H(9ґ), H(9)); 7.08—7.31 (m, 3 H, СНarom).
13C NMR (CD3OD), δ: 13.9 (MeBu N); 20.7 (CH2MeBu N);
4
22.7 (MeCON); 24.7 (CH2CH2MeB4u N); 40.5 (C(3)), 54.1
(С(5)), 59.4 (CH2NBu N), 64.2 (C(9))4, 69.5, 70.1, 73.1, 74.8
(C(4), C(6), C(7), C(84)), 104.2 (C(2)), 112.6 (СНarom), 113.6
(Сarom), 118.5 (СНarom), 121.4, 125.2 (Сarom), 126.6 (СНarom),
133.0, 134.6 (Сarom), 173.8 (C(1)), 175.6 (С=О). MS (positive
ion mode), found: m/z 242.2837 [Bu4N]+; C16H36N; calculatꢀ
ed: 242.2842. MS (negative ion mode), found: m/z 535.0125
[M – H]–; C19H21BrClN2O9; calculated: 535.0124.
1ꢀAcetylꢀ5ꢀbromoꢀ4ꢀchloroindolinꢀ3ꢀone (8). The reagent
80% H2SO4 (25 mL) was added to 3ꢀacetoxyꢀ1ꢀacetylꢀ5ꢀbroꢀ
moꢀ4ꢀchloroindole (4)29 (2.037 g, 6.16 mmol). The resulting
mixture was stirred at 20 °С for 1 h and poured into an ice—
water mixture. A precipitate formed was collected by filtration,
washed with 0.1 М NaOAc until washings had pH 7, and then
with Н2О. The target derivative 8 (1.5488 g, 87%) was obtained
after drying in vacuo over P2O5, which according to the NMR
data existed in the ketoꢀform. Rf 0.67 (chloroform—acetone,
1
(40—63 μm, Merck). H and 13C NMR spectra were recorded
on a Bruker ACꢀ200 (200.13 and 50.32, respectively) spectroꢀ
1
meter. H NMR chemical shifts are given relative to the residꢀ
ual signal of СHCl3 (δ 7.27) or relative to the signal of
H
CD2НOD (δ 3.31), 13C NMR shifts are given relative to the
Н
signal of СDCl3 (δ 77.0) or relative to the signal of CD3OD
C
(δ 49.0). The signals in the NMR spectra were assigned using
C
COSY and DEPTꢀ135 experiments. Mass spectra (electrоspray
ionization, ESI) were recorded on a Bruker micrOTOF II inꢀ
strument for 2•10–5 M solutions in MeCN. Optical rotation
was measured on a PUꢀ07 automatic digital polarimeter (Russia)
at 25 °C.
1
4 : 1). H NMR (CD3OD), δ: 2.31 (s, 3 H, CH3CON); 4.33
(s, 2 H, CH2N); 7.77 (d, 1 H, CHCHCBr, J = 8.3 Hz); 8.35
(d, 1 H, CHCBr, J = 8.3 Hz). 13C NMR (CD3OD), δ: 24.2
(CH3CON), 56.4 (CH2N), 117.6 (CHCHCBr), 140.7 (CHCBr),
118.8, 168.1, 190.6, 193.8 (Сarom). The NMR spectra are in
good agreement with those reported in the literature.29
Methyl (1ꢀacetylꢀ5ꢀbromoꢀ4ꢀchloroindolꢀ3ꢀylꢀ4,7,8,9ꢀtetraꢀ
Oꢀacetylꢀ5ꢀdiacetylaminoꢀ3,5ꢀdideoxyꢀαꢀDꢀglyceroꢀDꢀgalactoꢀ
nonꢀ2ꢀulopyranosid)onate (9), methyl (5ꢀbromoꢀ4ꢀchloroindolꢀ
3ꢀylꢀ4,7,8,9ꢀtetraꢀOꢀacetylꢀ5ꢀdiacetylaminoꢀ3,5ꢀdideoxyꢀαꢀDꢀ
glyceroꢀDꢀgalactoꢀnonꢀ2ꢀulopyranosid)onate (10), and methyl
(1ꢀacetylꢀ5ꢀbromoꢀ4ꢀchloroindolꢀ3ꢀylꢀ5ꢀacetamidoꢀ4,7,8,9ꢀ
tetraꢀOꢀacetylꢀ3,5ꢀdideoxyꢀαꢀDꢀglyceroꢀDꢀgalactoꢀnonꢀ2ꢀuloꢀ
Experiments on dynamic light scattering were carried out
on an ALVꢀCGSꢀ6010 goniometerꢀcorrelator (Langen, Gerꢀ
many) at a scattering angle of 150° using a Pyrex cell 10 mm in
diameter and a He—Ne laser (wavelength of 632.8 nm, 23 mW
power) as a source of radiation (more detailed information on
the experimental procedure and processing of the results is givꢀ
en in the works45,57). The samples were prepared by the addiꢀ
tion of anhydrous ethyl acetate (2 mL) or ethyl acetate saturatꢀ
ed with water (2 mL) to a weighed amount of sialyl chloride
(0.172 mmol). Prior to transfer to the cell, the resulting soluꢀ
tions were filtered (4 times) through a membrane filter (0.45 μm,
PTFE, Supelco). The samples were thermostated in a Lauda
thermostat at a temperature of 25.0 0.2 °С.
(5ꢀBromoꢀ4ꢀchloroindolꢀ3ꢀyl) (5ꢀacetamidoꢀ3,5ꢀdideoxyꢀαꢀ
DꢀglyceroꢀDꢀgalactoꢀnonꢀ2ꢀulopyranosid)onic acid, tetrabutylꢀ
ammonium salt (2). NꢀAcetylindoxyl derivative 8 (136.8 mg,
0.474 mmol), Bu4NHSO4 (291.6 mg, 0.860 mmol), and then
1 М aqueous solution of Na2CO3 (5 mL) were added a solution
of N,Nꢀdiacetylsialyl chloride 728 (236 mg, 0.428 mmol) in
anhydrous ethyl acetate (5 mL) under argon. After stirring for
21 h at room temperature (~20 °С), the reaction mixture was
diluted with ethyl acetate (25 mL). The organic phase was
washed with saturated aqueous NaHCO3 (2×50 mL) and satuꢀ
rated aqueous NaCl (50 mL), filtered through a cotton wool plug.
The solvent was evaporated on a rotary evaporator, Et3N
(0.6 mL) and H2O (0.9 mL) were added to a solution of the
* Although deacetylation was carried out in the presence of
triethylamine, triethylammonium salt of the acid was not isoꢀ
lated. Most likely, the tetrabutylammonium cation present in
the reaction mixture efficiently competes with other cations in
the formation of salts (note that triethylamine exists in unproꢀ
tonated form under basic deacetylation conditions) or is poorly
replaced by other cations (a wellꢀknown phenomenon). Apparꢀ
ently, in the course of chromatography on silica gel, partial ion
exchange of tetrabutylammonium cations with protons takes
place (silica gel is a weak acid). Unfortunately, we were not
able to completely replace tetrabutylammonium cations with
protons using cationites in the H form in protic solvents: in
all the cases, a cleavage of glycosidic bond and elimination
of aglycon with the formation of colored indigoꢀlike products
took place.

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Orlova et al.
pyranosid)onate (11). NꢀAcetylindoxyl derivative 8 (131 mg,
0.454 mmol), Bu4NHSO4 (315.5 mg, 0.931 mmol), and then
1 М aqueous solution of Na2CO3 (5 mL)were added to a solution
of N,Nꢀdiacetylsialyl chloride 7 (253 mg, 0.458 mmol) in anhyꢀ
drous ethyl acetate (5 mL) under argon. After stirring for 26 h
at room temperature (~20 °С), the reaction mixture was diluted
with ethyl acetate (25 mL). The organic phase was washed with
saturated aqueous NaHCO3 (2×50 mL) and saturated aqueous
NaCl (50 mL), filtered through cotton wool plug. The solvent
was evaporated on a rotary evaporator. The residue was disꢀ
solved in toluene (2 mL) and separated by gelꢀchromatography
on a column (50×2.5 cm) with BioꢀBeads SX3 (200—400 mesh,
BioꢀRad), using toluene as the eluent and a differential refracꢀ
tometer (Knauer) as a detector. A fraction, which, according to
the selective detection on TLC, contained glycosylation prodꢀ
ucts was collected (162.4 mg). Several fractions containing
glycosylation products with different degree of deacetylation
were isolated from this fraction by chromatography on silica
gel (gradient: toluene→toluene—acetone (7 : 3)).
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Fraction А (85.9 mg) contained (1H NMR data) indolyl
sialoside 9 as the mixture with N,Nꢀdiacetylglycal 1230 in the
ratio of 3.5 : 1. The yields of 9 (56.1 mg, 15%) and 12 (15.7 mg,
6%) were calculated by the integration of the corresponding
signals in the 1H NMR spectrum, assuming the molecular
weights of 9 and 12 being equal to 804.0 and 515.5, respectively.
Fraction B (9.3 mg, 1.2%) contained (1H NMR data) indolyl
sialoside 10. Fraction C (6.5 mg) contained (1H NMR data)
indolyl sialoside 11 as the mixture with Nꢀacetylglycal 1327,31,32 in
the ratio of 3.6 : 1. The yields of 11 (5.4 mg, 1.5%) and 13 (1.1 mg,
0.5%) were calculated by the integration of the corresponding
signals in the 1H NMR spectrum, assuming the molecular
weights of 11 and 13 being equal to 762.0 and 473.4, respectively.
1
Compound 9. Rf 0.50 (toluene—acetone, 7 : 3). H NMR
(CDCl3), δ (selected signals): 3.02 (dd, 1 H, Heq(3), J = 13.2 Hz,
J = 5.4 Hz); 3.65 (s, 3 H, OMe); 5.44—5.66 (m, 1 H, H(4));
7.52 (d, 1 H, СНarom, J = 9.3 Hz); 8.27 (d, 1 H, СНarom, J = 9.3 Hz).
13C NMR (CDCl3), δ (selected signals): 20.9 (CH3CO), 23.7
(MeCON), 100.8 (C(2)).
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b:rucb.0000035663.15439.84).
Compound 10. Rf 0.35 (toluene—acetone, 7 : 3). [α]D21 +25
(с 0.6, CHCl3). 1H NMR (CDCl3), δ: 1.91—2.48 (m, 19 H,
6 MeCO, Hax(3)); 3.08 (dd, 1 H, Heq(3), J = 12.9 Hz, J = 5.1 Hz);
3.62 (s, 3 Н, OMe); 3.98—4.37 (m, 3 H, Н(5), Н(6), Н(9а));
5.04—5.36 (m, 3 H, H(7), H(8), Н(9b)); 5.47—5.69 (m, 1 H,
H(4)), 6.88—7.53 (m, 3 H, СНarom); 8.19 (br.s, 1 Н, NH).
1
Compound 11. Rf 0.19 (toluene—acetone, 7 : 3). H NMR
(CDCl3), δ (selected signals): 2.34 (dd ~ t, 1 H, Hax(3),
J = 12.7 Hz); 2.92 (dd, 1 H, Heq(3), J = 12.7 Hz, J = 4.6 Hz);
3.62 (s, 3 Н, OMe); 7.56 (d, 1 H, СНarom, J = 8.8 Hz); 8.27
(d, 1 H, СНarom, J = 8.8 Hz).
This work was financially supported by the Russian
Science Foundation (Project No. 16ꢀ13ꢀ10244).
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Received October 5, 2017;
in revised form October 25, 2017