The use of the novel glycosyl acceptor and supramer analysis in the synthesis of sialyl-α(2–3)-galactose building block

Article abstract of DOI:10.1016/j.carres.2018.10.001

A new glycosyl acceptor to be used in sialylation was designed as a 3-hydroxy derivative of 4-methoxyphenyl β-D-galactopyranoside with 2-O-acetyl group and O-4 and O-6 protected as benzylidene acetal. Two alternative syntheses of this compound were compar

Full text of DOI:10.1016/j.carres.2018.10.001

Accepted Manuscript
The use of the novel glycosyl acceptor and supramer analysis in the synthesis of
sialyl-α(2–3)-galactose building block
Marina O. Nagornaya, Anna V. Orlova, Elena V. Stepanova, Alexander I. Zinin,
Tatiana V. Laptinskaya, Leonid O. Kononov
PII:
S0008-6215(18)30537-8
10.1016/j.carres.2018.10.001
CAR 7611
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 15 September 2018
Revised Date: 2 October 2018
Accepted Date: 2 October 2018
Please cite this article as: M.O. Nagornaya, A.V. Orlova, E.V. Stepanova, A.I. Zinin, T.V. Laptinskaya,
L.O. Kononov, The use of the novel glycosyl acceptor and supramer analysis in the synthesis of
sialyl-α(2–3)-galactose building block, Carbohydrate Research (2018), doi: https://doi.org/10.1016/
j.carres.2018.10.001.
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The use of the novel glycosyl acceptor and supramer analysis in the synthesis of sialyl-α(2-
3)-galactose building block
Marina O. Nagornaya a,b, Anna V. Orlova b, Elena V. Stepanova b,c, Alexander I. Zinin b, Tatiana
V. Laptinskaya d, Leonid O. Kononov a,*,1
a School of Advanced Manufacturing Technologies, National Research Tomsk Polytechnic
University, Lenin Avenue 30, Tomsk 634050, Russian Federation
b N. D. Zelinsky Institute of Organic Chemistry of the Russian Academy of Sciences, Leninsky
prosp., 47, 119991 Moscow, Russian Federation
c Research School of Chemistry & Applied Biomedical Sciences, National Research Tomsk
Polytechnic University, Lenin Avenue 30, Tomsk 634050, Russian Federation
d Faculty of Physics, M. V. Lomonosov Moscow State University, Leninskie Gory, 119992
Moscow, Russian Federation
Abstract
A new glycosyl acceptor to be used in sialylation was designed as a 3-hydroxy derivative of 4-
methoxyphenyl β-D-galactopyranoside with 2-O-acetyl group and O-4 and O-6 protected as
benzylidene acetal. Two alternative syntheses of this compound were compared. Sialylation of 3-
OH group of the glycosyl acceptor with O-chloroacetylated N-trifluoroacetylneuraminic acid
phenyl thioglycoside (NIS, TfOH, MeCN, MS 3 Å, –40 °C) was studied in a wide concentration
range (2–150 mmol·L–1). The outcome of sialylation generally followed the predictions of
supramer analysis of solutions of sialyl donor in MeCN, which was performed by polarimetry
and static light scattering and revealed two concentration ranges differing in solution structure
and the structures of supramers of glycosyl donor. The optimized conditions of sialylation (C
=50 mmol·L–1) were used to synthesize protected Neu-α(2-3)-Gal disaccharide (78%, α:β =
13:1), which was then converted to sialyl-α(2-3)-galactose imidate building block useful for the
synthesis of complex sialo-oligosaccharides.
Keywords: Neuraminic acid; Glycosylation; Sialylation; Concentration; Reactivity; Supramer
approach
1 Corresponding author. E-mail addresses: Leonid.Kononov@gmail.com, kononov@ioc.ac.ru (L.O. Kononov).
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1. Introduction
Sialic acid-containing glycoconjugates are ubiquitously and abundantly present at the
surface of all cell types in various organisms and involved in a wide range of biological
phenomena ranging from cell–cell adhesion and mobility to recognition by viruses and bacteria
[1-7]. Tremendous efforts have been made in order to develop efficient methods for
stereoselective synthesis of α-sialosides by chemical glycosylation (sialylation), and, therefore,
more efficient assembly of sialo-oligosaccharides and sialo-conjugates. Unfortunately, reliable
introduction of sialic acid residues into oligosaccharides remains quite a difficult problem and
poor predictability and reproducibility of yield and stereoselectivity are still typical of the
sialylation reaction (for leading references see the latest reviews [8-10]; a comprehensive list of
references can be found in a recent publication [11]). For this reason, a detour is commonly used
that during sialo-oligosaccharide assembly utilizes disaccharide building blocks, which contain
sialic acid residue at the non-reducing end and are prepared either by chemical or enzymatic
methods (see [12-26] and references cited therein). The main advantage of such an approach is
the minimization of a number of difficult sialylation steps. Since the sialyl-α(2-3)-galactose
disaccharide fragment is often found at the non-reducing end of sialo-glycans, especially
appealing is the use of sialyl-α(2-3)-galactose [Neu-α(2-3)-Gal] building blocks with removable
protective group at the N-5 of sialic acid residue which can be used in the divergent synthesis of
various N-acyl (including N-acetyl and N-glycolyl) derivatives of sialo-oligosaccharides with the
terminal sialic acid residue connected to O-3 of galactose [13-15, 18-22, 24-26].
Clearly, for the preparation of sialyl-α(2-3)-galactose block two monosaccharide units are
required: a sialyl donor and a galactosyl acceptor. As in other glycosylations [27], each
combination of sialyl donor and glycosyl acceptor, which in case of sialylation may have one or
several (up to four) hydroxy groups [28], is unique, the outcome of sialylation depending on
many variables [11, 29-34] including the nature of protective groups on both partners [22, 23,
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35] and concentration of reagents [30, 32, 33, 36]. While most studies on sialylation extensively
optimize the nature of glycosyl donors, only limited attention has been given to the design of
efficient nucleophiles for use in sialylation reactions. Not only should the reactivity of a glycosyl
acceptor match that of glycosyl donor [37], but, for practical reasons, the former also needs to be
preparatively accessible in good overall yield, preferably in minimal number of steps.
In this communication, we describe a short synthesis of a novel readily available glycosyl
acceptor with one hydroxy group at C-3 of galactose, demonstrate its utility in sialylation
reaction and accomplish an efficient synthesis of the sialyl-α(2-3)-galactosyl imidate building
block.
2. Results and discussion
2.1. Synthesis of glycosyl acceptor
2.1.1. Design of glycosyl acceptor
The new glycosyl acceptor was designed as a 3-hydroxy derivative of 4-methoxyphenyl β-
D-galactopyranoside 6 with 2-O-acetyl group and O-4 and O-6 protected as benzylidene acetal.
Galactose-based glycosyl acceptors with 4,6-O-benzylidene groups have already been used in
sialylation [14, 15, 20, 38]. Unlike benzoyl group, acetyl group only mildly deactivates the
vicinal hydroxy group,2,3 and we expected glycosyl acceptor 6 to be reactive enough to be
readily glycosylated with conventional sialyl donors rather than with more sophisticated sialyl
donors [20, 44-46] that often require very low temperatures. The presence of participating 2-O-
2 For a discussion on this issue, see [39]. Benzoyl group is more electron-withdrawing than acetyl group since
benzoic acid is somewhat more acidic (pKa 4.17) than acetic acid (pKa 4.76). This can lead to decreased
nucleophilicity of the vicinal hydroxy group at C-3.
3 Note that all neighboring acyl groups (including acetyl group at O-2) deactivate 3-OH of galactose often leading to
diminished yields of silaylation [40]. For this reason, the use of O-benzylated glycosyl acceptors in sialylation is a
more common practice [38, 41-43]. This issue is rather complex and a balanced discussion of the influence of the
nature and position of protective groups in glycosyl acceptor on the outcome of sialylation is clearly beyond the
scope of this publication.
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acetyl group would ensure 1,2-trans-stereochemistry in subsequent glycosylations with
prospective sialyl-α(2-3)-galactose building block [12-14, 16, 18, 19]. 4-Methoxyphenyl aglycon
has been used to protect anomeric position in glycosyl acceptor during sialylation step [25, 32]
and then cleaved under oxidative conditions to give hemiacetal thus allowing transformation to a
variety of disaccharide glycosyl donors [18, 21].
2-O-Substituted 4,6-O-benzylidene-β-D-galactopyranosides cannot be prepared by
4
selective protection of O-2 in 4,6-O-benzylidene-protected 2,3-diequatorial diols . A feasible
approach to such derivatives can rely on introduction of benzylidene group into a 3,4,6-triol with
the desired protective group at O-2.
Scheme 1 here
2.1.2. Preparation of 2-O-acetyl derivative 5 in one step by selective deacetylation
We have recently reported that 2-O-acetyl aryl glucopyranosides can be prepared in one-
step from per-O-acetylated aryl glycosides with β-gluco-configuration by selective O-
deacetylation under acidic conditions [48, 49]. This method is also applicable for aryl glycosides
with β-galacto-configuration [49]. Indeed, acid-catalyzed deacetylation (aq HCl, EtOH, CHCl3)
[49] of the known 4-methoxyphenyl 2,3,4,6 tetra-O-acetyl-β-D-galactopyranoside (1) [50]
readily afforded previously unknown 2-O-acetyl galactoside 5 in a good yield (55%) (Scheme 1)
along with tetraol 2 [50, 51] (40%), which can be re-acetylated to give 1 and then recycled in the
deacetylation reaction thus making this route to 5 even more attractive.
2.1.3. Preparation of 2-O-acetyl derivative 5 in four steps via acetal 4
4 Note that migration of acyl groups from O-3 to O-2 under basic conditions can be used for the preparation of 2-O-
acyl-4,6-O-benzylidene-β-D-galactopyranose derivatives [47].
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For comparison, a more traditional approach to 2-O-substituted β-D-galactopyranosides
was also explored. A reaction of tetraol 2 [50, 51], prepared from tetraacetate 1 [50] by Zemplén
deacetylation [52], with 2,2-dimethoxypropane in the presence of CSA under conditions of
thermodynamic control [53, 54] gave the known isopropylidene acetal 3 [55, 56] with free
hydroxy group at C-2, which was further acetylated to give fully protected derivative 4.
Cleavage of acetal groups with trifluoroacetic acid (TFA) [56] gave 2-O-acetyl glycoside 5
(Scheme 1) identical to that prepared by one-step procedure (see section 2.1.2). Although the
four-step procedure gave slightly higher overall yield of the target compound 5 (63%) it requires
much more time (more than 5 days) and effort in comparison to the one-step synthesis described
in section 2.1.2.
2.1.4. Preparation of glycosyl acceptor 6
The prepared galactoside 5 with one acetyl group at O-2 then was converted by reaction
with PhCH(OMe)2 in the presence of TsOH in MeCN to 4,6-O-benzylidene derivative 6 (83%)
(Scheme 1) that had only one free hydroxy group at C-3 making it a prospective glycosyl
acceptor in various glycosylation reactions. Importantly, alcohol 6 is highly soluble in MeCN
commonly used for sialylation thus making this compound suitable as glycosyl acceptor5 that
was demonstrated in this study (see section 2.2).
2.2. Sialylation
2.2.1. Design of sialyl donor
5 Note that although the corresponding derivative with benzoyl group at O-2 is known [47], it has never been used as
glycosyl acceptor in sialylation to the best of our knowledge. Similar 2-O-benzoyl-4,6-O-benzylidene derivatives of
galactose with different aglycons have been used as glycosyl acceptors in sialylation [20, 46].
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As discussed in section 1, a “good” sialyl donor should contain a cleavable protective
group at the N-5 of sialic acid residue that would allow subsequent introduction of the desired N-
acyl substituent (e.g., N-acetyl or N-glycolyl). Sialyl donors with N-trifluoroacetyl (TFA)
protective group have successfully been used in a number of syntheses of naturally occurring
sialyl derivatives (see [11] and references cited therein). For this reason, N-TFA-sialyl donor 7
[11] with O-chloroacetyl (CA) groups was chosen for testing ability of galactose derivative 6 to
act as glycosyl acceptor (Scheme 2). Since glycosylation of primary hydroxy group of 1,2:3,4-di-
O-isopropylidene-α-D-galactopyranose with sialyl donor 7 gave high yield (86%) and good
stereoselectivity (α:β = 11:1) [11] it was reasonable to test this sialyl donor in sialylation of a
secondary hydroxy group of derivative 6 (it is known that outcome of sialylation with the same
glycosyl donor may dramatically depend on the nature of glycosyl acceptor [8-10]).
Scheme 2 here
2.2.2. Supramer analysis of solutions of sialyl donor
2.2.2.1. Basics of the supramer approach
Since concentration of reagents is known to affect glycosylation outcome, one has to
choose concentrations of glycosyl donor and glycosyl acceptor. This can be done either by
analogy to previous examples of similar reactions or using some rationalization that could
suggest possibly optimal conditions, the latter approach being more appealing. For this reason,
before performing actual glycosylation experiments we studied, like previously [30, 32, 33, 36],
solutions of sialyl donor 7 in MeCN6 with different concentrations by polarimetry [34, 57, 58]7
and light scattering [32, 34, 59].8 This so called supramer analysis [36] of solutions is an integral
6 Sialylation reaction was performed in MeCN. See Experimental (section 4.6) for details.
7 Polarimetry is extremely sensitive to changes in solution structure (see [36] the detailed discussion).
8 Glycosyl donor 7 has earlier been used in sialylation only at a single “regular” concentration (50 mmol·L–1) [11],
its solutions have never been studied by supramer analysis.
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part of an approach, recently proposed by us [11, 29-36, 57-65] (see [33, 34] for the reviews),
which explicitly accounts for the structure of a reaction solution and is based on the hypothesis
that in many cases the real reactive species in solution are non-covalently-bonded
supramolecular aggregates, supramers, rather than isolated molecules of reagents.9 Changes in
solute concentration may influence structure of the corresponding supramers in a step-wise
manner [34, 36]. The concentration ranges, where supramers of similar structures hence
chemical properties exist, are separated by critical concentrations from other concentration
ranges, where differently arranged supramers featured by altered chemical properties (reactivity,
selectivity) are formed [34, 36]. The supramer approach was shown to be useful for explanation,
prediction and discovery of a series of unexpected phenomena and allowed the development of
highly efficient and stereoselective glycosylation reactions with sialyl donors that lead to
formation of Neu-α(2-3)-Gal [32, 33] and Neu-α(2-6)-Gal [31, 33] glycosidic linkages found in
many natural sialo-oligosaccharides of biological and medical significance.
2.2.2.2. Measurement of optical rotation of solutions of sialyl donor
Analysis of concentration dependence of specific optical rotation (SR, [α]D) of freshly
prepared solutions of sialyl donor 7 in MeCN (Fig. 1, a) revealed the existence of two ranges of
concentrations: high concentration range (C = 25–150 mmol·L–1), where the SR values almost
do not depend on concentration ([α]D = –(130–135) deg·dm–1·cm3·g–1), and low concentration
range (C = 2–5 mmol·L–1), where the SR values are noticeably different ([α]D = –(145–147)
deg·dm–1·cm3·g–1). The SR values measured at 10 mmol·L–1 were scattered and for this reason
this concentration cannot be unambiguously included in neither range (see error bars in Fig. 1,
a); it should be rather considered as a border-line concentration between the two ranges, i.e.
critical concentration.
9 For the detailed discussion of the foundations of the supramer approach and supramer analysis of solutions as well
as the relevant references not cited here see [36].
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According to the supramer approach (section 2.2.2.1), which associates the observed
differences in SR values of solutions with changes in solution structure (see [36] for the details),
this observation suggests two different types of supramers to be present in the high (C = 25–150
mmol·L–1) and low (C = 2–5 mmol·L–1) concentration ranges. Basing on previous experience
[32, 36], we may expect two different reactivity patterns in these two concentration ranges. Only
the experiment may reveal, in which concentration range sialyl donor 7 would perform better
both in terms of the product yield and sialylation stereoselectivity (see section 2.2.3).
Figure 1 here
2.2.2.3. Measurement of light scattering of solutions of sialyl donor
Our previous studies [32, 57, 59] suggest that in some cases different types of supramers,
which exist in different concentration ranges (as follows from supramer analysis of solutions by
polarimetry, see section 2.2.2.2), can be distinguished also by static or dynamic light scattering
(SLS and DLS, respectively). Analysis of intensities of scattered light (SLS) in solutions of sialyl
donor 7 was performed in terms of solvent quality, which is an approach normally used for
polymers [66-69] and was reported for analysis of structure of solutions of a low-molecular-mass
substance [57]. The measured values of scattering intensities were used for construction of the
Debye plot (Fig. 1, b) in which the slope (equal to the second virial coefficient A2) [57, 66, 67]
indicates the thermodynamic quality of solvent.
Interestingly, the solvent quality changes dramatically with concentration of sialyl donor 7
in MeCN (Fig. 1, b). Thus, for solutions with concentrations in the low concentration range (C =
2–5 mmol·L–1) the second virial coefficient A2 < 0 (negative slope) suggesting features of poor
solvent characterized by attraction between the solute particles leading to increased solute
aggregation and compaction of supramers, whereas for solutions with concentrations in the high
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concentration range (C = 25–150 mmol·L–1) A2 ~ 0 (horizontal line) typical of theta (θ) solvent
suggesting that different, less dense, supramers of the solute are formed in this range (assuming
the generalizations established for solutions of polymers are applicable in this case too). It is
important that these changes from poor to theta solvent correlate well with changes in SR values
as the critical point in Fig. 1, b (at ca. 10–25 mmol·L–1) corresponds to that in Fig. 1, a (at 10
mmol·L–1). Even more important is the finding that the high concentration range (C = 25–150
mmol·L–1) corresponds to theta solvent. This means that aggregation and solvation effects are
balanced (ideal conditions) and we can expect featureless glycosylation in the high concentration
range (C = 25–150 mmol·L–1). In other words, glycosylation performed in this concentration
range is expected to provide high yield of disaccharide10 independently of concentration used.11
At the same time, glycosylation in the low concentration range (C = 2–5 mmol·L–1) is expected
to be low-yielding as the solute molecules are expected to form tight supramers (with limited
accessibility of the molecules of glycosyl donor 7 located in the supramer core) similar to dense
collapsed polymer globules usually formed in poor solvents.
2.2.3. Results of sialylation experiments at different concentrations
Having established the existence of two concentration ranges featured by the presence of
different supramers of glycosyl donor, we performed a series of glycosylation experiments of
with equimolar amounts of sialyl donor 7 and glycosyl acceptor 6 (Scheme 2) under standard
conditions (NIS, TfOH, MeCN, MS 3 Å, –40 °C) at various concentrations that belong to these
ranges (Table 1, Fig. 1, c).12 In accordance with the results of supramer analysis of solutions of
10 Since the molecules of glycosyl donor 7, incorporated in relatively loose supramers that are formed under theta
conditions, are accessible for other reagents.
11 One may speculate that under theta conditions the outcome of glycosylation (including stereoselectivity) would be
determined mainly by molecular structure of reagents and follow generalization already found [8-10, 27], while
solution structure effects would play minimal role.
12 We intentionally used equimolar amounts of glycosyl donor 7 and glycosyl acceptor 6 since this experimental
design allows easy monitoring the reaction course and correct estimation of time required for the reaction to
complete. The use of excess of a sialyl donor is quite a common practice; in such cases, higher yields of
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sialyl donor 7 in MeCN (see section 2.2.2), the outcome of sialylation differed considerably
when the sialylation reaction was performed at concentrations belonging to the different
concentrations ranges. Not surprisingly, the yield of disaccharide 8 formed at concentrations that
belong to the high concentration range (C = 25–150 mmol·L–1), i.e. under theta conditions, was
high (68–78%) and almost did not change with concentration; all reactions were finished within
1 h (Table 1, Fig. 1, c). On the contrary, sialylation at concentration 5 mmol·L–1, which belongs
to the low concentration range (C = 2–5 mmol·L–1),13 i.e. in poor solvent, was sluggish and gave
much lower yield of disaccharide 8 (31%) after 28 h at –40 °C. This remarkable retardation of
glycosylation in dilute solution14 is in accordance (Fig. 1) with prediction made based on the
results of supramer analysis of solutions of sialyl donor 7 in MeCN by SLS (see section 2.2.2.3),
which suggests that molecules of glycosyl donor 7 located in the core of tight supramers, which
are formed in dilute solutions (C = 2–5 mmol·L–1) (see section 2.2.2.3), have limited
accessibility hence cannot efficiently participate in glycosylation reaction.
Stereoselectivity of sialylation was good (α:β = 9:1 – 13:1) in all cases studied (Table 1)
although the α/β ratio of disaccharide 8 had no clear relation to the concentration unlike
previously studied examples [32, 36]. Pure α- and β-isomers of disaccharide α-8 and β-8 were
isolated by silica gel chromatography and extensively characterized (see section 4.6).
Table 1 here
2.2.4. Comparison of results of supramer analysis and glycosylation experiments with
previously reported results
glycosylation products are usually obtained in line with general consensus that the competing elimination from a
sialyl donor is the main reason for diminished yields in sialylation.
13 Only one concentration from this range was used as an example. Practically useful glycosylations are normally
performed at reasonably high concentrations (50–500 mmol·L–1). Glycosylations in very dilute solutions, although
sometimes reported to be advantageous (see [36] for relevant references), are impractical since exceptionally large
volumes of solvent are required when the reactions are performed at the millimolar scale.
14 Similar observations have been reported [32, 36].
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Since this is the third example of the use supramer analysis for rational selection of
concentrations at which glycosylation is performed, it is reasonable to compare these three cases.
Although sialylation with two different glycosyl donors in MeCN was studied only in two (Ref.
[32] and this study) of three cases (the third one was arabinofuranosylation in 1,2-dichloroethane
[36]), it clear that there are obvious similarities as well as differences.
In all three cases, the glycosylation yields were virtually identical at concentrations greater
than a critical concentration found by polarimetry. At these critical concentrations, a plot of SR
of solutions of a glycosyl donor (in the solvent used for glycosylation) against concentration
experienced a discontinuity – either a minimum [32] or levelling off (Ref. [36] and this study).
According to the supramer approach (see section 2.2.2.2, [36, 57] and references cited therein)
identical SR values (levelling off at concentrations exceeding critical values) at different
concentrations (for the same compound dissolved in the same solvent) suggest similar structures
of supramers present at these concentrations, hence similar chemical properties of the solute. For
this reason, in these cases (Ref. [36] and this study) we limited ourselves to the study of solutions
of glycosyl donor only.15 However, in the case of a minimum of SR [32] this reasoning was no
longer valid. We could no longer ignore the presence of glycosyl acceptor in the reaction mixture
and performed additional experiments although this required extra efforts. Using polarimetry and
DLS for equimolar mixtures of glycosyl donor and glycosyl acceptor dissolved in MeCN, we
were able to reveal formation of hetero-supramers that incorporated the molecules of both
glycosyl donor and glycosyl acceptor [32]. Notably, these hetero-supramers were present only at
concentrations above the mentioned critical concentration, which was detected for solutions of
glycosyl donor, i.e. in the concentration range where glycosylation yield did not depend on
concentration. Interestingly, in all three cases studied glycosylation yield significantly dropped
while the reaction time increased in the low-concentration range (below the critical
15 Recall that the main aim of using supramer analysis is the rational selection of concentrations for glycosylation. In
the mentioned cases the choice of the concentrations was based on data for solutions of glycosyl donor only. As
experience suggests this simplified approach can provide effective means for optimization of glycosylation while
saving unnecessary efforts required for a more thorough study.
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concentration) suggesting lower reactivity of supramers of glycosyl donor formed at these
concentrations.
A possibility to use light scattering for studying solutions requires sufficient intensity of
scattered light (SLS and DLS) and adequate contrast (difference of refractive indices) between
the dispersed particles and the medium (DLS). Unfortunately, these conditions are not always
met.16 It is for this reason, we were able to use light scattering only in two of three cases. We
were lucky to use DLS in the first studied case [32] to get information on the size of supramers
present in reaction solutions. In this study, however, while optical contrast was small preventing
observation of a correlation function of scattered light (DLS), the intensity of scattered light was
sufficient for reliable SLS experiments (see section 2.2.2.3). These experiments for the first time
allowed us to gain insight into the inner structure of supramers, discriminating tight and loose
supramers that differed in their reactivity (see sections 2.2.2.3 and 2.2.3).
To summarize, the use of supramer analysis of solutions of either glycosyl donor or
mixtures of glycosyl donor and acceptor (in the reaction solvent) can provide a rational basis for
selection of concentrations at which glycosylation is performed.
2.3. Preparation of the sialyl-α(2-3)-galactose imidate building block
The obtained sialyl-α(2-3)-galactose disaccharide block α-8 has one obvious zest: it can
be further functionalized at galactose O-4 or O-6 after selective removal of benzylidene group
(see compound 10 in Scheme 3)17 to give a variety of more complex building blocks which can
be then elaborated in a way similar that described below.18 However, in the present study we
elected to convert pure disaccharide α-8 to a simpler sialyl-α(2-3)-galactose N-
16 These features of light scattering make polarimetry a method of choice for supramer analysis of reaction solutions.
17 For example, galactose 4,6-diols can be used as glycosyl acceptors.
18 This direction of research is currently being explored in our laboratories.
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phenyltrifluoroacetimidate building block 14 since imidates are widely used for the preparation
of oligosaccharides [70] (Scheme 3). Cleavage of benzylidene group under acidic conditions
gave diol 10, all O-acyl groups were removed by treatment with methanolic MeONa to give
heptaol 11, which was acetylated to give heptaacetate 12 (63% over 3 steps) identical to that
described by us earlier [18]. Methoxyphenyl anomeric protective group was then oxidatively
cleaved from O-acetylated disaccharide 12 to give hemiacetal 13 and imidoyl group was
successively introduced to obtain the target sialyl-α(2-3)-galactose imidate building block 14
(66% over 2 steps).
3. Conclusions
In conclusion, a new glycosyl acceptor to be used in sialylation was designed as a 3-
hydroxy derivative of 4-methoxyphenyl β-D-galactopyranoside 6 with 2-O-acetyl group and O-4
and O-6 protected as benzylidene acetal. Two alternative syntheses of compound 6 were
compared. Sialylation of 3-OH group of the glycosyl acceptor with O-chloroacetylated N-
trifluoroacetylneuraminic acid phenyl thioglycoside 7 (NIS, TfOH, MeCN, MS 3 Å, –40 °C) was
studied in a wide concentration range (2–150 mmol·L–1). The outcome of sialylation generally
followed the predictions of supramer analysis of solutions of sialyl donor 7 in MeCN, which was
performed by polarimetry and static light scattering and revealed two concentration ranges
differing in solution structure and the structures of supramers of glycosyl donor 7. The optimized
conditions of sialylation (C =50 mmol·L–1) were used to synthesize protected Neu-α(2-3)-Gal
disaccharide 8 (78%, α:β = 13:1), which was then converted to sialyl-α(2-3)-galactose imidate
building block 14 useful for the synthesis of complex sialo-oligosaccharides.
4. Experimental
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4.1. General Methods
Glycosylation reactions were carried out under argon atmosphere with anhydrous solvents.
The reactions were performed with the use of commercial reagents (Aldrich, Fluka, Acros
Organics). Anhydrous solvents were purified and dried (where appropriate) according to
standard procedures. Ethanol used for partial deacetylation of compound 1 contained 5% of
water. MeCN for glycosylation reactions was distilled under argon over P2O5 and then over
CaH2 and stored over molecular sieves (MS) 3 Å under argon. Powdered MS 3 Å (Fluka) were
activated before the reactions by heating at 220 °C in high vacuum for 24 h and then for 6 h just
before use in glycosylation. Gel permeation chromatography was performed in toluene on a
column (570 × 25 mm) packed with Bio Beads S-X3 gel (200–400 mesh, Bio-Rad) using a
differential refractive index detector (Knauer). Column chromatography was performed on silica
gel 60 (40–63 µm, Merck). Thin-layer chromatography was carried out on silica gel 60 F254
plates on aluminum foil (Merck). Spots of compounds were visualized under UV light (254 nm)
and by heating the plates (at ca. 150 °C) after immersion in a 1:10 (v/v) mixture of 85% aqueous
H3PO4 and 95% EtOH. HPLC analysis was carried out on an Agilent Compact LC instrument
equipped with a 150 × 4.6 mm Eclipse Plus C-18 (5 µm) column eluted with a gradient of MeCN
(from 0% to 100%) in H2O (that contained 0.1% trifluoroacetic acid) in 20 min at 0.4 mL/min
1
flowrate and UV detection (220 nm); sample volume was 20 µL. H, 13C NMR spectra were
registered for solutions in CDCl3 or CD3OD on a Bruker AM-300 instrument (300.13 and 75.48
MHz for 1H and 13C respectively) or on a Bruker AVANCE 600 spectrometer (600.13 and 150.9
1
1
MHz for H and 13C, respectively). The H NMR chemical shifts are referred to the residual
signal of CHCl3 (δH 7.27 ppm), CHD2OD (δH 3.31 ppm), the 13C NMR shifts – to the central line
of CDCl3 signal (δC 77.00 ppm), CD3OD signal (δC 49.00 ppm). Assignments of the signals in
the NMR spectra were performed using 2D-spectroscopy (COSY, HSQC, and HMBC) and
DEPT-135 experiments. High resolution mass spectra (electrospray ionization, HRESIMS) were
14

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recorded in a positive ion mode on a Bruker micrOTOF II mass spectrometer for 2 × 10–5 M
solutions in MeCN. Optical rotations were measured using a JASCO P-2000 automatic digital
polarimeter (Japan).
4.2. Experimental procedure for optical rotation measurements
Optical rotation values were measured with a Jasco P-2000 automatic digital polarimeter at
20 °C in a jacketed glass cell (10 cm length). Special precautions were made to ensure the
stability of the instrument and the temperature within the measuring compartment of the
instrument and the cell, which was maintained with an accuracy of ±0.1 °C. After the instrument
was warmed up for at least 1 h (as experience suggests, after this period the temperature within
the instrument remains stable for at least 8–10 h of continuous work) the instrument readings
were verified against the quartz standards (α = +21.267 and –21.248).
When studying the concentration dependence of the specific rotation, three solutions were
independently prepared for each concentration. Samples were prepared by dissolving the
weighed amount of compound 7 in anhydrous MeCN using a 2 mL (or 10 mL for concentration
2 mmol·L–1) volumetric flask immediately before the measurements. The solutions were filtered
through a 0.45 µm filter (PTFE, diameter 0.13 mm, Chromafil, Macherey Nagel; separate filters
were used for different samples) directly to the measuring cell with a jacket connected a Huber
СС-K6 (Exclusive) thermostat. Only after stabilization of temperature of the solution in the cell
(typically after 10 min; judged by the readings of built-in Pt100 temperature probe and
stabilization of the values of optical rotation) sampling of the data began. The optical rotation
was measured continuously (TimeCourse program supplied the Jasco P-2000 polarimeter) for 30
min (1800 sample points with 1 s integration time each). The data obtained were averaged and
the corresponding mean value and standard deviation were calculated for each concentration.
These data are plotted in Fig. 1, a as specific rotation values that were calculated using
concentrations (c) of the solutions expressed in traditional polarimetric units (g/100 mL) unless
15

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otherwise explicitly stated; in Fig. 1 molar concentrations are shown, which are identical to those
indicated in Table 1.
4.3. Experimental procedure for light scattering measurements
For static light scattering (SLS) experiments, three solutions of compound 7 in anhydrous
MeCN were independently prepared for each concentration as described in section 4.2
immediately before measurements. Each sample was filtered three times through a 0.45 µm
filter, (PTFE, diameter 0.13 mm, Chromafil, Macherey Nagel; separate filters were used for
different samples). SLS measurements were performed with an ALV Correlation Goniometer
System 5000/6010 (Langen, Germany) at 150° scattering angle using Pyrex cells (1 cm in
diameter) and a He–Ne laser (632.8 nm, 23 mW) as the light source. The temperature of the
scattering cell was maintained at 20 ± 0.2 °C. Intensities of scattered light were measured by
photon counting and are expressed in Hertz. For each solution, scattering intensities were
averaged over 20 independent measurements (the total collection time was 20 min for each
experiment). Solvent scattering I0 (I0 = 7.5 ± 0.1 kHz) was subtracted from total solution
scattering I to obtain the excess intensity of scattered light (I – I0). Then the resulting three
values for each concentration were averaged and the corresponding mean value and standard
deviation were calculated for each concentration. These data were used to construct a Debye plot
(Fig. 1, b) in which the slope (equal to the second virial coefficient A2) [66, 67] indicates the
thermodynamic quality of solvent (in the polymer sense) [57, 66-69].
4.4. 4-Methoxyphenyl 2-O-acetyl-β-D-galactopyranoside (5)
4.4.1. Preparation in one step
Crystalline 4-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1) [50] (454
mg, 1 mmol) was dissolved in CHCl3 (1 mL) and 95% EtOH (3 mL) was added followed by
16

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36% HCl (1 mL, d = 1.18 g·mL–1). The homogenous reaction mixture was kept at 30 °C for 10 h
until HPLC analysis showed that the peak of the target monoacetate 5 (tR = 9.8 min) became the
–
major one. Anion-exchange resin Amberlite AB-17 (HCO3 ) was added until pH 7 was achieved
then filtered off, the resin was washed with 95% EtOH (20 mL) and the combined filtrate was
concentrated under reduced pressure to give the residue that was purified by silica gel column
chromatography (gradient CHCl3–95% EtOH, 9:1 → 3:1) to give 4-methoxyphenyl-β-D-
galactopyranoside (2) [50, 51] as white crystals (114 mg, 40%), which could be recycled, and the
target triol 5 obtained as white crystals after crystallization from EtOAc–petroleum ether (180
mg, 55%).
4.4.2. Preparation in four steps via acetal 4
Crystalline 4-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1) [50] (1.59
g, 3.5 mmol) was suspended in anhydrous MeOH and 0.1 M methanolic MeONa (5 µL, 0.5
µmol) was added and the mixture was stirred at ~20 °C for 18 h, then neutralized with cation-
exchange resin Amberlyst 15 (H+). The resin was filtered off, washed with MeOH (20 mL) and
the combined filtrate was concentrated under reduced pressure, the residue was dried in vacuo to
give crude tetraol 2 [50, 51] (1 g, ~100%; Rf = 0.38, CHCl3–EtOH, 4:1). A mixture of tetraol 2
(1 g, 3.5 mmol) and (1S)-(+)-camphorsulfonic acid (17.5 mg, 75 mmol) was dried in vacuo for 2
h and 2,2-dimethoxypropane (105 mL, 850 mmol) was added under argon [53, 54]. The reaction
mixture was stirred at ~20 °C for 3 days and then quenched with Et3N (7 mL, 0.05 mol), the
mixture was stirred for 30 min, concentrated under reduced pressure, toluene was added and then
concentrated (2 × 10 mL). The residue, which contained mainly compound 3 [55, 56] (Rf = 0.67,
CHCl3–EtOAc, 5:1), was dissolved in anhydrous pyridine (4 mL, 0.05 mol) followed by addition
of Ac2O (8 mL, 0.085 mol) and stirred at ~20 °C for 38 h. Methanol (8 mL) was added, the
mixture was stirred for 15 min, concentrated under reduced pressure, toluene was added and then
concentrated (2 × 10 mL). The residue, which contained mainly compound 4 (Rf = 0.33,
17

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petroleum-ether–EtOAc, 7:3), was dissolved in CH2Cl2 (20 mL) and the solution was cooled to 0
°C (ice–water bath) and a 10:1 (v/v) mixture of trifluoroacetic acid–water (3.3 mL) was added.
The reaction mixture was stirred at ~20 °C for 1 h until TLC showed full conversion of the
starting 4 (Rf = 0.90, CHCl3–EtOH, 5:1) to a lower running spot (Rf = 0.50, CHCl3–EtOH, 5:1)
and then concentrated under reduced pressure, toluene was added and then concentrated (2 × 10
mL). The residue was purified by silica gel column chromatography (gradient CHCl3–95%
EtOH, 9:1 → 3:1) to give triol 5 as a white solid (0.723 g, 63% over three steps).
4.4.3. Data for 4-methoxyphenyl 2-O-acetyl-
β
-D-galactopyranoside (5)
24
Mp 124–125 °C (EtOAc–petroleum ether). [α]D + 11.8 (c 1.0, 95% EtOH). Rf = 0.50
1
(CHCl3–95% EtOH, 5:1). H NMR (300 MHz, CD3OD, δ, ppm, J, Hz): 2.10 (s, 3H, CH3CO),
3.68 (ddd, 1H, J4,5 1.0, J5,6a 5.1, J5,6b 6.8, H-5), 3.74 (s, 3H, C6H4OCH3), 3.75 (dd, 1H, J3,4 3.5,
J2,3 9.9, H-3), 3.77 (dd, 1H, J5,6a 5.1, J6a,6b 11.3, H-6a), 3.82 (dd, 1H, J5,6b 6.8, J6a,6b 11.3, H-6b),
3.94 (dd, 1H, J4,5 1.0, J3,4 3.5, H-4), 4.89 (d, 1H, J1,2 8.0, H-1), 5.25 (dd, 1H, J1,2 8.0, J2,3 9.9, H-
13
2), 6.80–6.88 (m, 2H, OC6H4O), 6.92–7.01 (m, 2H, OC6H4O). C NMR (75 MHz, CD3OD, δ,
ppm): 21.0 (CH3CO), 56.1 (C6H4OCH3), 62.3 (C-6), 70.4 (C-4), 73.1 (C-3), 73.8 (C-2), 77.1 (C-
5), 102.1 (C-1), 115.6, 119.2, 153.0, 156.8 (OC6H4O), 172.2 (CH3CO). HRESIMS: found m/z
367.0788 [M + K]+. Calcd forC15H20O8K: 367.0790.
4.5. 4-Methoxyphenyl 4,6-O-benzylidene-2-O-acetyl-β-D-galactopyranoside (6)
4-Methoxyphenyl 2-O-acetyl-β-D-galactopyranoside (5) (0.895 g, 2.7 mmol) was
dissolved in anhydrous MeCN (100 mL) and p-toluenesulphonic acid monohydrate (23 mg, 0.12
mmol) was added followed by PhCH(OMe)2 (809 µL, 5.4 mmol). The reaction mixture was
stirred at ~20 °C for 18 h, Et3N (50 µL, 0.36 mmol) was then added and the reaction mixture was
concentrated under reduced pressure. Purification of the residue by silica gel column
chromatography (gradient toluene–95% EtOH, 15:1 → 3:1) gave the title compound 6 as a white
18

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24
solid (0.944 g, 83%). Mp 104–106 °C. [α]D –28.4 (c 0.97, CHCl3). Rf = 0.48 (toluene–95%
EtOH, 9:1). 1H NMR (300 MHz, CDCl3, δ, ppm, J, Hz): 2.15 (s, 3H, CH3CO), 2.60 (d, 1H, JOH-
11.0, OH-3), 3.57 (ddd, 1H, J4,5 0.6, J5,6b 1.3, J5,6a 1.6, H-5), 3.78 (s, 3H,C6H4OCH3), 3.81
3,3
(ddd, 1H, J3,4 3.7, J2,3 9.9, J3-OH,3 11.0, H-3), 4.10 (dd, 1H, J5,6a 1.6, J6a,6b 12.4, H-6a), 4.26 (dd,
1H, J4,5 0.6, J3,4 3.7, H-4), 4.37 (dd, 1H, J5,6b 1.3, J6a,6b 12.4, H-6b), 4.90 (d, 1H, J1,2 8.0, H-1),
5.33 (dd, 1H, J1,2 8.0, J2,3 9.9, H-2), 5.57 (s, 1H, CHPh), 6.78–6.88 (m, 2H, OC6H4O), 6.97–7.06
13
(m, 2H, OC6H4O), 7.35–7.44 (m, 3H, Ph), 7.54 (dd, 2H, J 3.1, J 6.6, Ph). C NMR (75 MHz,
CDCl3, δ, ppm): 20.9 (CH3CO), 55.6 (C6H4OCH3), 66.7 (C-5), 68.9 (C-6), 71.7 (C-3), 72.0 (C-
2), 75.4 (C-4), 100.6 (C-1), 101.5 (CHPh), 114.5, 119.0 (OC6H4O), 126.5, 128.3, 129.3, 137.3
(Ph), 151.3, 155.6 (OC6H4O), 170.4 (CH3CO). HRESIMS: found m/z 439.1362 [M + Na]+.
Calcd for C22H24O8Na: 439.1303.
4.6. Typical glycosylation procedure
A mixture of thoglycoside sialyl donor 7 [11] (77.5 mg, 0.1 mmol) and alcohol 6 (41.6 mg,
0.1 mmol) was dried in vacuo for 2 h, then anhydrous MeCN (see Table 1 for the concentrations
used) was added under argon. Freshly activated powdered MS 3 Å (Fluka; 100 mg per 1 mL of
MeCN) were added to the resulting solution. The suspension was stirred under argon at ~20 °C
overnight (15 h), then cooled to –40 °C (MeCN–solid CO2 bath). Solid NIS (53.4 mg, 0.3 mmol)
(dried in vacuo for 2 h) was added under argon followed by neat TfOH (2 µL, 0.02 mmol).
Persistent iodine color became visible in 1–2 min. The reaction mixture was stirred under argon
at –40 °C until TLC showed complete consumption (see Table 1 for reaction times) of the
starting thioglycoside 7 (Rf = 0.78, toluene–acetone, 5:1).19 Saturated aq NaHCO3 (2 mL) was
added and the reaction mixture was diluted with CH2Cl2 (20 mL), stirred for 5 min, filtered
through Celite pad, the solids were additionally washed with CH2Cl2 (100 mL). The combined
19 Complete consumption of the starting thioglycoside 7 (40–60 min for experiments performed at 50–150 mmol·L–1
concentrations, entries 2–5 in Table 1) was accompanied by appearance of green color of the reaction mixture. Note
that reaction performed in dilute solution (5 mmol·L–1, entry 1 in Table 1) did not turn green, iodine color persisted.
19

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filtrate was washed with satd aq NaHCO3 (50 mL), 1.2 M aq Na2S2O3 (2 × 50 mL), water (2 ×
50 mL), dried over Na2SO4, filtered through a cotton wool plug, concentrated under reduced
pressure, toluene was added and then concentrated (2 × 10 mL). The residue was dried in vacuo,
dissolved in toluene (2 mL) and separated by gel permeation chromatography on Bio-Beads S-
X3 (toluene). The first eluted fraction contained disaccharide 8, which was analyzed by NMR
spectroscopy to give anomeric ratio values (α:β, see Table 1; for determination of ratio of
anomers of disaccharide 8 integral intensities of signals of α-H-3eq and β-H-3eq of sialic acid
residue were used). The disaccharide fraction was chromatographed on a silica gel 60 column
(gradient toluene → toluene–acetone, 5:1) to give pure α- and β-isomers of disaccharide 8 as
white solids. For the yields obtained at different concentrations, see Table 1 and Fig. 1, c.
4.6.1. Data for methyl [4-methoxyphenyl 2-O-acetyl-4,6-O-benzylidene-3-O-(4,7,8,9-tetra-
O-chloroacetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-β-D-galacto-nonulopyranosyl)-β-
D-galactopyranosid]onate ( -8).
α
[α]D24 + 4.9 (c 1.0, CHCl3). Rf = 0.30 (toluene–acetone, 5:1). 1H NMR (600 MHz, CDCl3,
δ, ppm, J, Hz): 1.88 (dd~t, 1H, J3eq,3ax= J3ax,4 12.8, H-3axII), 2.21 (s, 3H, CH3CO), 2.84 (dd, 1H,
J3eq,4 4.6, J3eq,3ax 12.8, H-3eqII), 3.67 (ddd~tq, 1H, J4,5 1.0, J5,6a=J5,6b 1.5, H-5I), 3.72 (s,
3H,CO2CH3), 3.78 (s, 3H, C6H4OCH3), 3.86 (dd, 1H, J4,5 1.0, J3,4 3.7, H-4I), 3.97 (d, 1H, J 14.9,
CH2Cl), 4.01 (d, 1H, J 14.9, CH2Cl), 4.02 (s, 2H, CH2Cl), 4.09 (dd, 1H, J5,6a 1.5, J6a,6b 12.4, H-
6aI), 4.11 (d, 1H, J 14.1, CH2Cl), 4.12–4.15 (m, 2H, H-5II, H-6II), 4.14 (dd, 1H, J8,9a 5.8, J9a,9b
12.7, H-9aII), 4.15 (d, 1H, J 14.1, CH2Cl), 4.21 (d, 1H, J 15.7, CH2Cl), 4.35 (dd, 1H, J5,6b 1.5,
J6a,6b 12.4, H-6bI), 4.45 (d, 1H, J 15.7, CH2Cl), 4.46 (dd, 2H, J3,4 3.7, J2,3 10.1, H-3I), 4.54 (dd,
1H, J8,9b 2.4, J9a,9b 12.7, H-9bII), 5.00 (ddd, 1H, J3eq,4 4.6, J3ax,4 12.8, J4,5 9.8, H-4II), 5.09 (d, 1H,
J1,2 8.0, H-1I), 5.39 (s, 1H, CHPh), 5.42 (dd, 2H, J6,7 2.4, J7,8 9.5, H-7II), 5.42 (dd, 2H, J1,2 8.0,
J2,3 10.1, H-2I), 5.72 (ddd, 1H, J8,9b 2.4, J8,9a 5.8, J7,8 9.5, H-8II), 6.64 (d, 1H, J5,NH 9.1, NH),
6.81–6.85 (m, 2H, OC6H4O), 7.01–7.06 (m, 2H, OC6H4O), 7.33–7.41 (m, 3H, Ph), 7.51 (dd, 2H,
20

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13
J 1.7, J 7.6, Ph). C NMR (151 MHz, CDCl3, δ, ppm): 21.1 (COCH3), 38.0 (C-3II), 40.2, 40.2,
40.5, 41.6 (CH2Cl), 49.8 (C-5II), 53.4 (CO2CH3), 55.7 (C6H4OCH3), 63.4 (C-9II), 66.1 (C-5I),
68.1 (C-2I), 68.7 (C-8II), 69.1 (C-6I), 69.3 (C-7II), 69.8 (C-4II), 71.4 (C-6II), 72.4 (C-3I), 73.5 (C-
4I), 96.8 (C-2II), 100.6 (C-1I), 101.1 (CHPh), 113.3 (q, J 287.3, CF3), 114.5, 119.0 (OC6H4O),
126.3, 128.2, 130.9, 137.5 (Ph), 151.4, 155.5 (OC6H4O), 158.0 (q, J 39.4, COCF3), 166.5, 167.0,
167.2, 167.4 (ClCH2CO), 168.7 (C-1II), 169.7 (CH3CO). HRESIMS: found m/z 1102.1155 [M +
Na]+. Calcd for C38H42F3NO16Na: 1102.1055.
4.6.2. Data for methyl [4-methoxyphenyl 2-O-acetyl-4,6-O-benzylidene-3-O-(4,7,8,9-tetra-
O-chloroacetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-β-D-galacto-nonulopyranosyl)-β-
D-galactopyranosid]onate ( -8).
β
[α]D29 + 14.3 (c 1.0, CHCl3). Rf = 0.45 (toluene–acetone, 5:1). 1H NMR (600 MHz, CDCl3,
δ, ppm, J, Hz): 1.90 (dd, 1H, J3ax,4 11.6, J3ax,3eq 13.0, H-3axII), 2.14 (s, 3H, CH3CO), 2.64 (dd,
1H, J3eq,4 4.7, J3eq,3ax 13.0, H-3eqII), 3.72 (ddd~dd, 1H, J5,6b 1.4, J5,6a 2.7, H-5I), 3.78 (s, 3H,
C6H4OCH3), 3.88 (s, 3H, CO2CH3), 3.92 (d, 1H, J 14.7, CH2Cl), 3.96 (d, 1H, J 14.7, CH2Cl),
4.04–4.09 (m, 2H, CH2Cl), 4.08–4.20 (m, 8H, C-3I, 2× CH2Cl, H-6aI, H-9aII, H-5II), 4.37 (dd,
2H, J5,6b 1.4, J6a,6b 12.7, H-6bI), 4.38 (dd, 1H, J6,7 2.7, J6,5 10.5, H-6II), 4.48 (dd, 1H, J4,5 0.9, J3,4
3.9, H-4I), 4.96 (d, 1H, J1,2 8.1, H-1I), 5.34 (dd, 1H, J7,8 2.0, J6,7 2.7, H-7II), 5.39 (ddd, 1H, J3eq,4
4.7, J4,5 10.4, J3ax,4 11.6, H-4II), 5.45 (dd, 1H, J8,9b 2.6, J9a,9b 12.4, H-9bII), 5.50 (dd, 1H, J1,2 8.1,
J2,3 10.0, H-2I), 5.58 (ddd, 1H, J7,8 2.0, J8,9b 2.6, J8,9a 9.5, H-8II), 5.70 (s, 1H, PhCH), 6.80–6.86
(m, 3H, OC6H4O, NH), 6.99–7.03 (m, 2H, OC6H4O), 7.41–7.49 (m, 3H, Ph), 7.60–7.63 (m, 2H,
13
Ph (H-2, H-6)). C NMR (151 MHz, CDCl3, δ, ppm, J, Hz): 20.9 (CH3CO), 37.9 (C-3II), 40.1,
40.4, 40.5, 40.6 (CH2Cl), 49.5 (C-5II), 53.2 (CO2CH3), 55.6 (C6H4OCH3), 63.8 (C-9II), 66.3 (C-
5I), 69.1 (C-6I), 69.3, 69.4 (C-2I, C-4II), 70.8 (C-7II), 72.4 (C-6II), 73.6 (C-8II), 74.9 (C- (C-2, C-
6)), 75.5 (C-3I), 100.3 (C-2II), 100.5 (C-1I), 101.1 (PhCH), 114.5, 119.1 (OC6H4O), 126.2 (Ph
(C-2, C-6)), 128.7 (Ph (C-3, C-5)), 129.8 (Ph (C-4)), 137.5 (Ph (C-1)), 151.2 (OC6H4O (C-1)),
21

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155.7 (OC6H4O (C-4)), 158.0 (q, J 37.4, CF3CO), 166.0 (C-1II), 166.4, 167.0, 167.6, 167.8
(ClCH2CO), 169.4 (CH3CO).20 HRESIMS: found m/z 1102.1121 [M + Na]+. Calcd for
C38H42F3NO16Na: 1102.1055.
4.7. Methyl [4-methoxyphenyl 2,4,6-tri-O-acetyl-3-O-(4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-trifluoroacetamido-D-glycero-α-D-galacto-nonulopyranosyl)-β-D-galactopyranosid]onate
(12).
A solution of α-isomer of disaccharide α-8 (53.8 mg, 0.05 mmol) in 80% aq AcOH (1
mL), was heated at 70 °C for 8 h until TLC showed full conversion of the starting α-8 (Rf 0.54,
CHCl3–95% EtOH, 9:1) to a lower running spot (10, Rf 0.50, CHCl3–95% EtOH, 9:1). The
reaction mixture was concentrated under reduced pressure, toluene was added and then
concentrated (2 × 10 mL) to give crude 10 (38.7 mg, 78%) which was used without further
purification.
Crude 10 (25 mg, 0.025 mmol) was dissolved in anhydrous MeOH (2 mL) and 1 M
methanolic MeONa (10 µL, 0.01 mmol) was added and the mixture was stirred at ~20 °C for 48
h. Then AcOH (10 µL) was added and the mixture was stirred for 15 min, then concentrated
under reduced pressure, toluene was added and then concentrated (2 × 10 mL) to give crude 11
(16.1 mg, 99%; Rf 0.47, CHCl3–MeOH, 3:1), which was used without further purification.
Crude 11 (15 mg, 0.023 mmol) was dissolved in anhydrous pyridine (200 µL, 2.49 mmol)
and Ac2O (100 µL, 1 mmol) and stirred at ~20 °C for 15 h. Methanol (100 µL) was added and
the mixture was stirred for 15 min, then concentrated under reduced pressure, toluene was added
and then concentrated (2 × 10 mL). The residue was purified by silica gel column
chromatography (gradient toluene– acetone, 9:1 → 3:1) to give fully O-acetylated disaccharide
24
12 as a white solid (18 mg, 82%; 63% over three steps). [α]D + 2.0 (c 1.0, СHCl3). Rf 0.44
1
(toluene–acetone, 5:1). H NMR (300 MHz, CDCl3, δ, ppm, J, Hz): 1.75 (dd~t, 1H, J3ax,3eq
=
20 The signal of CF3 group could not be detected due to low signal-to-noise ratio.
22

ACCEPTED MANUSCRIPT
J3ax,412.7, H-3axII), 1.96, 2.01, 2.06, 2.09, 2.13, 2.19, 2.25 (all s, 21H, CH3CO), 2.67 (dd, 1H,
J3ax,3eq12.7, J3eq,4 4.6, H-3eqII), 3.78 (s, 3H, C6H4OCH3), 3.83 (dd, 1H, J6,7 2.4, J5,6 10.7, H-6II),
3.88 (s, 3H, CO2CH3), 3.93–4.05 (m, 3H, H-5II, H-5I, H-9aII), 4.08–4.16 (m, 2H, H-6I), 4.38 (dd,
1H, J8,9b 2.0, J9a,9b 12.5, H-9bII), 4.68 (dd, 1H, J3,4 3.1, J2,3 10.0, H-3I), 4.99 (d, 1H, J3,4 3.1, H-
4I), 5.05 (ddd, 1H, J4,3eq 4.6, J4,5 10.6, J4,3ax 12.7, H-4II), 5.10 (d, 1H, J1,2 8.0, H-1I), 5.29 (dd, 1H,
J1,2 8.0, J2,3 10.0, H-2I), 5.35 (dd, 1H, J6,7 2.4, J7,8 9.0, H-7II), 5.56 (ddd~td, 1H, J8,9b 2.0, J7,8
=
J8,9a 9.0, H-8II), 6.43 (d, 1H, J5,NH 9.4, NH), 6.83 (d, 2H, J 9.0, OC6H4O), 7.01 (d, 2H, J 9.0,
OC6H4O).13C NMR (75 MHz, CDCl3, δ, ppm): 20.4, 20.6, 20.6 (2C), 20.7, 20.9, 21.4 (CH3CO),
37.4 (C-3II), 49.9 (C-5II), 53.3 (CO2CH3), 55.7 (C6H4OCH3), 62.0 (C-9II), 62.3 (C-6I), 67.0 (C-
4I), 67.5 (C7II), 67.9 (C-8II), 68.6 (C-4II), 69.7 (C-2I), 70.8 (C-3I), 71.3 (C-6II), 71.5 (C-3I), 96.9
(C-2II), 100.2 (C-1I), 114.5 (OC6H4O), 115.3 (q, J 288.3, CF3), 118.5 (OC6H4O), 151.4
(OC6H4O), 155.5 (OC6H4O), 157.7 (q, J 38.1, COCF3), 167.7 (C-1II), 169.4, 169.6, 170.3 (2C),
170.5, 170.6, 170.7 (CH3CO). HRESIMS: found m/z 962.2502 [M + Na]+. Calcd
forC39H48F3NO22Na: 962.2518.
4.8. Methyl [2,4,6-tri-O-acetyl-1-O-(N-phenyltrifluoroacetylimidoyl)- 3-O-(4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-α-D-galacto-nonulopyranosyl)-β-D-
galactopyranose]onate (12).
Solution of (NH4)2Ce(NO3)6 (197 mg, 0.361 mmol) in 4:1 (v/v) MeCN–H2O mixture (0.8
mL) was slowly added to a cold (0 °C, ice–water bath) solution of glycoside 12 (67.6 mg, 0.072
mmol) in 4:1 (v/v) MeCN–H2O mixture (1.5 mL) and then the reaction mixture was stirred at 0
°C for 1 h. The reaction mixture was poured into aq NaHCO3 (50 mL) and extracted with CH2Cl2
(2 × 50 mL). Combined extracts were washed with brine (50 mL), water (50 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure to give hemiacetal 13 (50.4 mg, 84%)
as an α/β-mixture which was used without further purification.
23

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Crude hemiacetal 13 (36 mg, 0.043 mmol) was dried in vacuo for 2 h, dissolved in CH2Cl2
(1.5 mL) then ClC(NPh)CF3 [71] (10.3 µL, 0.064 mmol) and Cs2CO3 (21 mg, 0.064 mmol) were
added at 0 °C (ice–water bath) under argon. The reaction mixture was stirred at 0 °C (ice–water
bath) for 1 h and at ~20 °C for 15 h until TLC showed full conversion of the starting 13 (Rf 0.42,
toluene–acetone, 5:1) to a higher running spot (Rf 0.36, toluene–acetone, 5:1) and then
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography (gradient toluene–acetone (+0.1% Et3N), 9:1 → 3:1; the eluent was made basic
by addition of Et3N (100 µL per 100 mL of eluent)) to give imidate β-14 as a white solid (34 mg,
25
1
78%; 66% over 2 steps). [α]D + 0.3 (c 0.96, CHCl3). Rf 0.36 (toluene–acetone, 5:1). H NMR
(600 MHz, CDCl3, δ, ppm, J, Hz): 1.75 (dd~t, 1H, J3ax,3eq = J3ax,4 12.7, H-3axII), 1.97, 2.02, 2.11,
2.14, 2.18, 2.26 (all s, 21H, CH3CO), 2.66 (dd, 1H, J3eq,4 4.7, J3ax,3eq 12.7, H-3eqII), 3.82 (dd, 1H,
J6,7 2.7, J5,6 10.7, H-6II), 3.89 (s, 3H, CO2CH3), 3.97–4.00 (m, 1H, H-5I), 4.00 (ddd~dd, 1H, J4,5
= J5,NH 10.1, J5,6 10.7, H-5II), 4.01 (dd, 1H, J8,9a 6.3, J9a,9b 12.4, H-9aII), 4.06 (dd, 1H, J5,6a 7.6,
J6a,6b 11.3, H-6aI), 4.12 (dd, 1H, J5,6b 5.8, J6a,6b 11.3, H-6bI), 4.43 (dd, 1H, J8,9b 2.6, J9a,9b 12.4, H-
9bII), 4.73 (dd, 1H, J3,4 3.4, J2,3 9.1, H-3I), 4.99 (d, 1H, J3,4 3.4, H-4I), 5.03 (ddd, 1H, J3eq,4 4.7,
J4,5 10.1, J3ax,4 12.7, H-4II), 5.29 (dd~t, 1H, J1,2 = J2,3 9.1, H-2I), 5.34 (dd, 1H, J6,7 2.7, J7,8 8.5, H-
7II), 5.57 (ddd, 1H, J8,9b 2.6, J8,9a 6.3, J7,8 8.5, H-8II), 5.85 (br.s, 1H, H-1I), 6.41 (d, 1H, J5,NH 10.1,
13
NH), 6.86 (d, 2H, J 7.7, Ph), 7.10–7.14 (m, 1H, Ph), 7.31 (dd, 2H, J 7.5, J 8.4, Ph). C NMR
(151 MHz, CDCl3, δ, ppm): 20.4, 20.6 (2C), 20.6, 20.7, 21.4 (CH3CO), 37.4 (C-3II), 49.9 (C-5II),
53.4 (CO2CH3), 61.6 (C-6I), 62.4 (C-9II), 67.1 (C-4I), 67.3 (C-7II), 68.1 (C-8II), 68.5 (C-4II), 68.8
(C-2I), 71.3 (C-3I), 71.5 (C-6II), 71.6 (C-5I), 95.1 (C-1I), 96.9 (C-2II), 119.4 (PhN (C-2, C-6)),
124.4 (PhN (C-4)), 128.7 (PhN (C-3, C-5)), 143.4 (PhN (C-1)), 157.7 (q, J 30.3, COCF3), 167.6
(C-1II), 169.3, 169.4, 170.2, 170.3, 170.6, 170.6, 170.7 (CH3CO).21 HRESIMS: found m/z
1027.2389 [M + Na]+. Calcd for C40H46F6N2O21Na: 1027.2392.
21 Signals of CF3C=N and CF3 groups could not be detected due to low signal-to-noise ratio.
24

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Acknowledgements
Authors are grateful to Dr. K. P. Birin for NMR spectra. This work was financially supported by
the Russian Science Foundation (Project No. 16-13-10244).
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://doi.org/10.1016/***.
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Scheme 1. Preparation of glycosyl acceptor 6 by two alternative routes. Reagents and conditions: a. MeONa,
MeOH, ~20 °C. b. Me2C(OMe)2, CSA, ~20 °C. c. Ac2O, Py, ~20 °C. d. TFA, H2O, CH2Cl2, 0 °C (63% over 4
steps). e. aq HCl, EtOH, CHCl3, 30 °C (55% of 5 and 40% of 2). f. PhCH(OMe)2, TsOH·H2O, MeCN, ~20 °C
(83%).
Scheme 2. Glycosylation of alcohol 6 with sialyl donor 7. Reagents and conditions: a. NIS, TfOH, MeCN, MS 3 Å,
–40 °C. See Table 1 for other conditions, yields of 8 and α/β ratios obtained.
28