Experimental and theoretical study of the O3/O4 regioselectivity of glycosylation reactions of glucopyranosyl acceptors

Article abstract of DOI:10.1016/j.tet.2020.131719

The knowledge of the regioselectivity between different hydroxyl groups of glycosyl acceptors is valuable in planning simple strategies for the synthesis of oligosaccharides, minimizing the use of protecting groups. With the aim of obtaining deeper knowle

Full text of DOI:10.1016/j.tet.2020.131719

Journal Pre-proof
Experimental and theoretical study of the O3/O4 regioselectivity of glycosylation
reactions of glucopyranosyl acceptors
Enrique A. Del Vigo, Carlos A. Stortz, Carla Marino
PII:
S0040-4020(20)30939-X
https://doi.org/10.1016/j.tet.2020.131719
TET 131719
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 August 2020
Revised Date: 16 October 2020
Accepted Date: 23 October 2020
Please cite this article as: Del Vigo EA, Stortz CA, Marino C, Experimental and theoretical study of
the O3/O4 regioselectivity of glycosylation reactions of glucopyranosyl acceptors, Tetrahedron, https://
doi.org/10.1016/j.tet.2020.131719.
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Experimental and theoretical study of the O3/O4
regioselectivity of glycosylation reactions of glucopyranosyl
acceptors
Enrique A. Del Vigo, Carlos A. Stortz, Carla Marino*
Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Consejo Nacional
de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Hidratos de
Carbono (CIHIDECAR). Departamento de Química Orgánica, Pab. 2, Ciudad
Universitaria, 1428 Buenos Aires, Argentina.
* Corresponding author:
Tel/fax +54 11 45763346, E-mail: cmarino@qo.fcen.uba.ar (C. Marino).
1

Abstract
The knowledge of the regioselectivity between different hydroxyl groups of
glycosyl acceptors is valuable in planning simple strategies for the synthesis of
oligosaccharides, minimizing the use of protecting groups. With the aim of obtaining
deeper knowledge on this subject, we analyzed the relative reactivity of the OH-3 and
OH-4 groups of 2,6-di-O-protected methyl α- and β-glucopyranosides in glycosylation
reactions. The glycosyl acceptors were prepared by simple procedures, and galacto-
pyranosyl and furanosyl trichloroacetimidates were evaluated as glycosyl donors.
Experimental results were contrasted with those obtained by a molecular modeling
approach. A fair agreement of the molecular modeling and experimental results was
obtained. It has been shown, that by choosing the right anomer and protecting group,
either the 1→3 or 1→4 linkage can selectively be installed using the appropriate glucosyl
acceptor.
2

Introduction
Nowadays, the importance of specific oligosaccharides in biological processes is
widely known, thus requiring simple and reproducible procedures for their synthesis.1,2
Glycosylation reactions are a key factor in this process, though they often appears to
complicate the synthesis due to the large number of free hydroxyl groups present in
carbohydrates, many times with similar reactivities. This fact frequently makes it necessary
to introduce protecting groups which have to be removed at the end, giving rise to a
tedious, time-consuming and yield-depleting synthetic sequence. However, it is sometimes
possible to plan selective glycosylations avoiding or minimizing the use of protecting
groups if the reactivity of one of the hydroxyl groups is much larger than that of other one
and limiting amounts of glycosyl donors are used. This approach requires comprehensive
knowledge of the regioselectivity of glycosidation reactions, which are not always well
understood, as they depend sometimes on subtle factors. Some attempts to rationalize the
regioselectivity on theoretical basis have been fairly success,3 although others have failed.
In a recent work, we analyzed the relative reactivity of the OH-3 and OH-4 of 2,6-O-
diprotected methyl α- and β-galactopyranosyl derivatives in glycosylation reactions with
different galactosyl donors. As expected, the formation of the 1→3 disaccharides was
favored, given the equatorial orientation of the OH-3 group vs. the axial one of the OH-4
group. Nevertheless, the regioselectivities were very different, ranging from very high to
quite low, depending on the substituents and the anomeric configuration of the acceptors,
and the reactivities of the donors. The highest regioselectivities were achieved for the 2,6-
di-O-benzylated
acceptors
and
the
per-O-benzoylated-galactopyranosyl
trichloroacetimidate.4 Nevertheless, for some donors derived from 2-phthalilamide-2-
deoxy-glucopyranose the best regioselectivity was observed for a 2,6-di-O-benzoylated
galactopuranosyl acceptor,5 showing once again that the regioselectivity of glycosylation
reactions is susceptible to a manifold of factors. Rationalization of the OH-3/OH-4
reactivities by different molecular modeling approaches agreed with the general trend but
failed to explain subtler factors governing the difference in regioselectivity between some
of the acceptors.4
3

The two most common monosaccharide residues in Nature are
galactose, and consequently, these units have been used by carbohydrate chemists most
frequently. Motifs -Galp-1→3- -Glcp and -Galp-1→4- -Glcp (lactose) are used as
D-glucose and D-
D
D
D
D
models for the action of β-galactosidases,6 whereas other disaccharides and higher
oligosaccharides containing these moieties can have important biological properties. Thus,
knowledge of the selectivity between O-3/O-4 of glucosyl acceptors is significant in
planning the synthesis of these oligosaccharides when intending to minimize the use of
protecting groups. Although many studies related with the regioselective glycosylation of
“less protected acceptors” have been reported,5,7–9 the case of glucose, with all equatorial
substituents, has been less attended, usually not involving the O-3/O-4 positions.10–14
The aim of the present work is to extend our previous study to the case of glucosyl
acceptors, analyzing the glycosylation of 2,6-di-O-protected methyl glycosides 1α,β and
2α,β, which differ from the previous study4 in the fact that both free OHs are equatorial
(Figure 1). Trichloroacemidates 3 and 4 were used as donors, and the experimental results
were compared with those obtained by molecular modeling. Differences with
acceptors are also analyzed.
D
-Gal
Figure 1. Studied glucosyl acceptors and galactosyl donors
4

1. Results and Discussion
1.1. Experimental study
For the objectives of the present study we required D-Glcp derivatives with OH-2 and
OH-6 blocked. Many efforts have been made to develop simple procedures for the
preparation of partially substituted derivatives of glucose,13,15–20 which is more difficult to
achieve, compared with mannose or galactose, due to the fact that all the secondary
hydroxyl groups are equatorial and display comparable reactivity under various common
conditions. We synthesized derivatives 1α,β and 2α,β, in order to evidence the possible
differences between the electron withdrawing/not withdrawing character of the protecting
groups in the glycosylation regioselectivity. α-Glucopyranosyl acceptors were synthesized
from commercially available methyl α-D-Glcp (5α). For the β-anomers, instead, methyl β-
D
-Glcp (5β)21 was prepared by BF3·OEt2 promoted glycosidation22 of per-O-benzoyl-Glcp
under short reaction time to prevent α-anomerization, followed by Zemplèn de-O-
benzoylation. Benzylated glycosides 1α17 and 1β18 were obtained by stannylene acetals-
mediated substitutions, by treatment of 5α or 5β with Bu2SnO under toluene reflux,
followed by benzylation with benzyl bromide in the presence of TBAB (Scheme 1). The
general procedure described by Zhou et al.18 was followed. Benzoylated acceptor
2α23,24 was prepared by benzoylation with limited amounts of benzoyl chloride. It is worth
mentioning, that this procedure was not effective for the β-anomer. Acceptor 2β16 was
prepared also by stannylene acetals-mediated substitutions, but treatment with Bu2SnO of
5β was performed under MeOH reflux, followed by benzoylation in chloroform, as
described by Dong et al.25 (Scheme 1). The regioselective substitutions proceeded by
formation of the 4,6- and 2,3-stannylene acetals which are then substituted at the most
reactive O-2 and O-6 possitions.18
5

Scheme 1. Synthesis of D-Glcp glycosyl acceptors 1α,β and 2α,β
With glucosyl acceptors 1α,β and 2α,β in hand, we assayed glycosylation reactions
with galactosyl donors 3 and 4, with participating groups at position 2, and thus giving rise
only to β glycosides. Trichloroacetimidates 526 and 627 were prepared by treatment with
trichloroacetonitrile and DBU of the corresponding benzoylated hemiacetals. To avoid
migration during the glycosylations, acetyl groups were not used either in the donors or the
acceptors.28,29 The coupling reactions were performed in CH2Cl2 solution, at low
temperature, and the activation of the donor was acchieved with catalytic TMSOTf. The
molar ratio of acceptor to donor was 1.4:1 to preclude double glycosylation of the
acceptors.4
When TLC showed the disappearance of the donor, normally after 2 h of reaction,
1
relative yields of the disaccharides were determined by integration of the H NMR signals
of the anomeric or other well-resolved protons of the crude mixtures. Then, the mixtures
were purified by column chromatography in order to characterize the condensation
products and to confirm the yields of the isolated regioisomers. The structures of the
disaccharides were univocally assigned on the basis of NMR spectra. The position of the
interglycosidic linkages was verified from the deshielding of the 13C NMR signals involved
in such linkages.30,31 For example, for disaccharide 6β, linked (1→3), the main product of
the coupling between 3 with 1β, signals corresponding to C-3 and C-4 were observed at
6

86.0 and 68.7 ppm, respectively. Instead, for the minor product 7β (1→4) such signals
were observed at 75.3 (C-3) and 81.4 ppm (C-4). The deshielding effect of the
glycosidation can be clearly observed in Table 1, where C-3 and C-4 chemical shifts (126
MHz, Cl3CD) of acceptors 1α,β and 2α,β and dissacharides 6-13 are listed, as well as the
∆δ values between the dissacharides and the corresponding acceptor and the difference
between the ∆δ of C-3 and C-4 (∆∆δ). ∆∆δ values are > 0 for 1→3 dissaccharides and < 0
1
for 1→4. Information provided by HMBC experiments and observation in the H NMR
spectrum of the signal corresponding to the free OH and the corresponding 3JH,OH coupling
constant, also supported the regiochemistry of the products. The stereochemistries of the
3
newly formed glycosidic linkages were established from the J1’,2’ coupling constants,
which were around 8 Hz for disaccharides obtained from pyranosic donor 3 and 1-2 Hz for
those obtained from furanosic donor 4.32
13
Table 1. C NMR chemical shifts for C-3 and C-4 of the glycosyl acceptors 1α,β and
2α,β and the product dissacharides 6-13; ∆δn: difference between δC-n οf each
disaccharide and δC-n of the corresponding acceptor; ∆∆δ: ∆δ3 − ∆δ4.
Acceptors
3α
3β
4α
4β
72.9
70.9
75.9
71.5
71.4
70.6
75.2
70.6
C-3 (δ)
C-4 (δ)
1→3 Disaccharides
6α
6β
8α
8β
10α
10β
12α
79.7
8.3
12β
83.0
7.8
84.2
11.3
68.5
-2.4
13.7
86.0
10.1
68.7
-2.8
12.9
82.0
10.6
69.3
-1.3
11.9
85.9 81.3 82.9
10.7 8.4 7.0
68.8 69.5 70.2
C-3 (δ)
Δδ3
69.5
-1.1
9.4
69.5
-1.1
8.9
C-4 (δ)
Δδ4
-1.8
-1.4
9.8
-1.3
8.3
12.5
ΔΔδ
1→4 Disaccharides
7α
7β
9α
9β
11α
11β
13α
13β
72.0
-0.9
75.0
-0.9
69.9
-1.5
73.5 71.9 74.9
-1.7 -1.0 -1.0
82.3 77.2 77.1
70.3
-1.1
73.9
-1.3
C-3 (δ)
Δδ3
81.4
10.5
81.5
10.0
82.7
12.1
79.8
9.2
79.5
8.9
C-4 (δ)
Δδ4
11.7
6.3
5.6
-11.4
-10.9
-13.6
-13.4
-7.3
-6.6 -10.3
-10.2
ΔΔδ
7

In the glycosylation reactions, all the acceptors showed the same trend, except
methyl 2,6-di-O-benzoyl-β- -Glcp (2β, Table 2). Acceptors 1α, 1β and 2α were
D
preferentially glycosylated at O-3 (Table 2, entries 1-3 and 5-7), although it has been
stated that both OHs have similar steric hindrance.33 It was observed a higher selectivity
for the benzylated derivative 1α than for the benzoylated derivative 2α. For 2β, instead,
the 1→4 disaccharide 9β was the main product with 3 as glycosyl donor (entry 4), and no
regioselectivity was observed hen donor 4 was utilized (entry 8). For donor 3, less
reactive than 4,34,35 higher regioselectivities were achieved (entries 1-4 vs- 5-8).
These results show that a trend increasing the reactivity of O-4 is observed when
passing from benzylated derivatives to benzoylated derivatives, and when passing from
α-anomers to β-anomers. This trend leads to O-4 preferential regioselectivity only for
compound 2β. Exactly the same trend was observed when assaying the O-3/O-4
regioselectivity of α-and β-methylglycosides of 2-deoxy-2-dimethylmaleoyl glucosamine
derivatives protected with benzoyl and benzyl groups at O-6.34 However, in that case, the
trend led to preferential O-4 reactivity for both β-anomers. The reversal of regioselectivity
observed in these cases shows that the remote anomeric configuration plays a significant
role in the O-3/O-4 regioselectivity.
It is worth noticing that the yield was excellent in all glycosylations, showing once
again the robustness of the trichloroacetimidate method.
Scheme 2. Glycosylation of D-Glcp acceptors 1α,β and 2α,β with donors 3 and 4.
8

Table 2. Ratios and yields of 1→3 and 1→4 disaccharides obtained by reaction of donors
3-5 with acceptors 3α,β and 4α,β
Entry
Donor
Acceptor
Products
Ratioa
Yieldsb
a
c
1
→
3
1
→
4
1
→
3 : 1
6.5:1
2:1
→
4
%
%
1
2
3
4
5
6
7
8
96
96
71
100
92
90
100
100
91
92
75
90
89
87
92
91
3
3
3
3
4
4
4
4
1α
1β
2α
2β
1α
1β
2α
2β
6α
6β
8α
8β
10α
10β
12α
12β
7α
7β
9α
9β
11α
11β
13α
13β
2.1:1
1:5.6
3.45:1
1.75:1
2:1
1:1
aDetermined from the 1H NMR spectrum of the crude reaction mixture.
bCombined yield of 1→3 and 1→4 regioisomers.
cYields refers to isolated pure products after column chromatography on the basis of the
donor amount used in the reaction.
2.2. Molecular modeling study
We have carried out molecular modeling calculations in order to determine the
atomic partial charges and condensed-to-atom Fukui functions36 in an attempt to
rationalize the observed reactivity of the OH-3/OH-4 groups of acceptors 1α,β and 2α,β,
as we did with the equivalent galactose acceptors.4 As OH-3 in galactose was equatorial
and OH-4 was axial, and both are equatorial in glucose, it could be expected a less sharp
trend shown by modeling. The charge density was calculated for both methods using the
regular Mulliken charges.
For the sake of simplicity, instead of acceptors 1α,β and 2α,β, analogs carrying
acetyl groups instead of benzoyl, and methyl groups instead of benzyl moieties were
used37 (Figure 2). The lower energy conformers were found by molecular mechanics
calculations (MM3), and then optimized with B3LYP/6-311+G**, and then, single-point
calculations with M06-2X/6-311+G** (Figure A.1 and Table A.1, Electronic
Supplementary Information File 1). A whole set of low-energy conformers was calculated
for each of the four compounds, in order to carry out a Boltzmann-averaging to obtain the
9

local charges and Fukui functions (Table A.2 and Table A.3, Electronic Supplementary
Information File 1). It has been shown that in several systems the electron density
distribution can define the reactivity, i.e. the position with a larger net charge (q) will be
that preferably attacked by a hard electrophiles.38 However, in order to describe the
interaction between nucleophile and electrophile corresponding to a soft–soft interaction
in the context of the hard and soft acid and base principle (HSAB), the most common
numerical descriptor is the local Fukui function (f-), related to the electron density in the
HOMO frontier molecular for an electrophilic attack.36,39–41 It should be expected to
obtain higher absolute values for q and for f for the more reactive site. Fukui functions
have already been successfully used in order to assess the reactivity of secondary
hydroxyl groups of carbohydrate.42–44
Figure 2 and Table 3 show that the charge and Fukui determinations predict that
OH-3 should be the more reactive site for the benzylated derivatives 1α and 1β, whereas
OH-4 should be the more reactive site for the benzoylated derivatives 2α and 2β. This
indicates, that the experimental reactivity for the benzylated derivatives is predicted
correctly by modeling. On the other hand, for benzoylated derivatives, modeling predicts
correctly the higher HO-4 reactivity of 2β, but fails to perform correctly for 2α. The
highest regioselectivity observed for 1α appears not to be predicted either by using
modeling, suggesting that subtle factors not considered in the charge determinations, or by
energy errors which might lead to an inadequate Boltzmann-averaging of the low-energy
conformers can have some influence on the final outcome. The q and f values calculated
for both oxygens are very similar, much more than in the case of
D
-Galp acceptors,4
making the prediction of the regioselectivity less reliable, given the low significance of
the differences. The recognized limitation of the DFT to reproduce the conformational
dynamics of flexible systems,45–49 and the influence of the donor structure, not considered
in the present study, but certainly important,50 could be some reasons for this divergence
with the experimental results for 1α. In any case, a fair agreement of the experimental
results and the modeling has been determined.
10

Figure 2. Model D-Glcp 3,4-diol acceptors and data obtained with B3LYP.
Table 3. Ratio of charges and Fukui functions on O-3/O-4 observed for analogs of
acceptors 1-2 (calculations with M06-2X/6-311+G**//B3LYP/6-311+G**)
a
qO-3/qO-4
fO-3/fO-4
Analog of 1α
Analog of 1β
Analog of 2α
Analog of 2β
1.006
1.079
0.898
0.962
1.12
1.54
0.93
0.87
2. Conclusions
Glucosyl acceptors 1α,β and 2α,β were obtained by simple procedures. For
alkylated acceptors 1α,β, a higher reactivity for OH-3 was experimentally observed in
accordance with the theoretical results. For acylated acceptors 2α and 2β modeling predicts
a slightly higher reactivity of OH-4 which only agrees with the experimental results for 2β.
A trend towards a relatively enhanced reactivity of O-4 with regards to O-3 in the changes
α → β and Bz → Bn was observed, in agreement with previous reports. The
regioselectivities experimentally achieved in some of the cases, could significantly simplify
11

the synthesis of
D-Galp-1→3-D-Glcp and D-Galp-1→4-D-Glcp motifs in the synthesis
relevant molecules.
3. Experimental section
4.1 General Methods
The solvents used were distilled, dried and stored according to standard procedures.
Analytical thin layer chromatography (TLC) was performed on Silica Gel 60 F254 (Merck)
aluminium supported plates (layer thickness 0.2 mm). Visualization of the spots was
effected by exposure to UV light and charring with a solution of 5% (v/v) sulfuric acid in
EtOH, containing 0.5% p-anisaldehyde. Column chromatography was carried out with
Silica Gel 60 (230–400 mesh, Merck). Optical rotations were measured with a Perkin-
Elmer 343 digital polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded
with a Bruker AMX 500 instrument. Chemical shifts (δ) are reported in ppm, with residual
1
13
1
chloroform (δ 7.26 for H and δ 77.16 for C) as internal references. Assignments of H
1
and 13C NMR spectra were assisted by 2D H COSY and HSQC experiments. High
resolution mass spectra (HRMS) were obtained by Electrospray Ionization (ESI) and Q-
TOF detection.
4.2. General procedure for glycosylations using trichloroacetimidates 5 or 6 as glycosyl
donors
Powdered 4 Å molecular sieves (0.5 g) were added to a solution of
trichloroacetimidate 526 or 627 (73 mg, 0.1 mmol) and glycosyl acceptor 1α,β or 2 α,β (0.14
mmol, 1.4 equiv) in anhyd. CH2Cl2 (8 mL), and the suspension was stirred under Ar for 30
min. Then, the mixture was cooled to −30 °C and TMSOTf (8 µL, 0.044 mmol) was added.
When TLC analysis showed optimal conversion (normally 2 h of stirring at −30°C) the
reaction mixture was quenched with triethylamine, filtered and coevaporated under reduced
pressure with toluene. The residue was purified by column chromatography, as indicated in
each case.
12

4.2.2. Methyl 2,3,4,6-tetra-O-benzoyl-
glucopyranoside (6 ) and methyl 2,3,4,6-tetra-O-benzoyl-
2,6-di-O-benzyl-a- -glucopyranoside (7
β
-
D
-galactopyranosyl-(1→3)-2,6-di-O-benzyl-
α
-
D
-
α
β-D
-galactopyranosyl-(1→4)-
D
α)
1
Obtained according to the general procedure by condensation of 3 with 1α. H
NMR spectrum of the crude mixture showed the presence of two regioisomers,
6α and 7α, in a 6.5:1 ratio according to the integration of the signals corresponding to H-3’
of both disaccharides (5.63 and 5.50 ppm). TLC analysis of the crude mixture showed,
among the excess of 1α, a single product of Rf 0.37 (9:1 toluene-EtOAc), which after
column chromatography purification (90:10→88:12 toluene/EtOAc) was resolved in two
components with similar Rf. The first fractions afforded a mixture of disaccharides enriched
1
in 7α (0.03 g, 28%). H NMR(500 MHz, CDCl3) signals for 7α: δ 8.15-7.18 (m, 30H,
aromatic), 5.95 (dd, 1H, J4',5' = 0.8 Hz, J3',4' = 3.5 Hz, H-4'), 5.80 (dd, 1H, J1',2' = 8.1 Hz, J2',3'
= 10.5 Hz, H-2'), 5.50 (dd, 1H, J3',4' = 3.5 Hz, J2',3' = 10.5 Hz, H-3'), 4.86 (d, H, Jgem = 12.4
Hz, CH2Ph), 4.74 (d, 1H, J1',2' = 8.1 Hz, H-1'), 4.63 (d, H, Jgem = 12.4 Hz, CH2Ph), 4.60 (dd,
1H, J5',6a' = 4.9 Hz, Jgem = 11.6 Hz, H-6’a), 4.54 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.53 (dd, 1H,
J5',6b' = 7.9 Hz, Jgem = 11.6 Hz, H-6’b), 4.35 (d, 1H, Jgem = 12.2 Hz, CH2Ph), 4.32 (ddd, 1H,
J4',5' = 0.8 Hz, J5',6a' = 4.9 Hz, J5',6b' = 7.9 Hz, H-5'), 4.13 (d, 1H, Jgem = 12.2 Hz, CH2Ph),
4.12 (ddd, 1H, J3,OH = 0.7 Hz, J3,4 = 8.1 Hz, J2,3 = 9.5 Hz, H-3), 4.03 (d, 1H, J3,OH = 0.7 Hz,
OH-3), 3.69 (dd, 1H, J3,4 = 8.1 Hz, J4,5 = 9.9 Hz, H-4), 3.65 (ddd, 1H, J5,6b = 1.6 Hz, J5,6a
=
3.2 Hz, J4,5 = 9.9 Hz, H-5), 3.47 (dd, 1H, J5,6a = 3.2 Hz, Jgem = 10.8 Hz, H-6a), 3.38 (dd,
1H, J1,2 = 3.6 Hz, J2,3 = 9.5 Hz, H-2), 3.35 (dd, 1H, J5,6b = 1.6 Hz, Jgem = 10.8 Hz, H-6b),
3.28 (s, 3H, CH3O) ppm. 13C NMR (126 MHz, CDCl3):
δ 101.6 (C-1'), 98.6 (C-1), 81.4 (C-
4), 78.3 (C-2), 73.6 (CH2Ph), 73.1 (CH2Ph), 72.0 (C-3), 71.9 (C-5'), 71.4 (C-3'), 69.6 (C-
2'), 68.4 (C-5), 67.9 (C-4'), 67.7 (C-6), 62.4 (C-6'), 55.3 (CH3O) ppm.
Further elution of the column afforded syrupy compound 6α (0.064 g, 63%), [α]D +65
1
(c 1, CHCl3). H NMR (500 MHz, CDCl3): δ 8.12-7.21 (m, 30H, aromatic), 5.99 (dd, 1H,
J4',5' = 0.8 Hz, J3',4' = 3.5 Hz, H-4'), 5.93 (dd, 1H, J1',2' = 8.1 Hz, J2',3' = 10.4 Hz, H-2'), 5.63
(dd, 1H, J3',4' = 3.5 Hz, J2',3' = 10.4 Hz, H-3'), 5.11 (d, 1H, J1',2' = 8.1 Hz, H-1'), 4.58 (dd, 1H,
J5',6a' = 5.1 Hz, Jgem = 11.7 Hz, H-6’a), 4.56 and 4.53 (2d, 2H, Jgem = 12.3 Hz, 2x CH2Ph),
4.52 (dd, 1H, J5',6b' = 7.7 Hz, Jgem = 11.7 Hz, H-6’b), 4.40 (ddd, 1H, J4',5' = 0.8 Hz, J5',6a'
=
5.1 Hz, J5',6b' = 7.7 Hz, H-5'), 4.31 (d, 1H, Jgem = 12.8 Hz, CH2Ph), 4.25 (d, 1H, J1,2 = 3.6
13

Hz, H-1), 4.01 (d, 1H, Jgem = 12.8 Hz, CH2Ph), 3.95 (dd, 1H, J3,4 = 8.1 Hz, J2,3 = 9.5 Hz, H-
3), 3.89 (br. s, 1H, OH-4), 3.74-3.59 (m, 4H, H-4, H-5, H-6a and H-6b), 3.37 (dd, 1H, J1,2 =
3.6 Hz, J2,3 = 9.5 Hz, H-2), 3.23 (s, 3H, CH3O) ppm. 13C NMR (126 MHz, CDCl3):
δ 165.9, 165.4, 165.3 (COPh), 138.2, 138.0, 133.6, 133.36, 133.31, 133.2, 129.9, 129.77,
129.71, 129.2, 129.0, 128.7, 128.6, 128.5, 128.38, 128.33, 128.27, 128.25, 128.23, 127.8,
127.6, 127.46, 127.45 (aromatic), 102.1 (C-1'), 98.0 (C-1), 84.2 (C-3), 77.9 (C-2), 73.6
(CH2Ph), 73.4 (CH2Ph), 71.8 (C-5'), 71.6 (C-3'), 70.3 (C-5), 69.6 (C-2'), 69.0 (C-6), 68.5
(C-4), 68.0 (C-4'), 62.2 (C-6'), 54.9 (CH3O) ppm. ESIMS: m/z calcd for C55H52NaO15
[M+Na]+ 975.3198. Found: 975.319547.
4.2.3. Methyl 2,3,4,6-tetra-O-benzoyl-
glucopyranoside (6 ) and methyl 2,3,4,6-tetra-O-benzoyl-
2,6-di-O-benzyl- -glucopyranoside (7
β
-
D
-galactopyranosyl-(1→3)-2,6-di-O-benzyl-
β-D-
β
β-D
-galactopyranosyl-(1→4)-
β
-
D
β)
Obtained according to the general procedure by condensation of 3 with 1β. After 2 h
TLC analysis showed total consumption of 3 and a single spot of Rf 0.47 (85:15 toluene-
1
EtOAc) composed, according to the H NMR spectrum of the crude mixture, by two
regioisomers in 2:1 ratio. The products could not be separated by column chromatography
(0.09 g; 91%) but all the NMR signals could be assigned.
The major product was identified as 6β. 1H NMR (500 MHz, CDCl3): δ 5.98 (dd, 1H,
J4',5' = 0.7 Hz, J3',4' = 3.5 Hz, H-4'), 5.88 (dd, 1H, J1',2' = 8.1 Hz, J2',3' = 10.5 Hz, H-2'), 5.62
(dd, 1H, J3',4' = 3.5 Hz, J2',3' = 10.5 Hz, H-3'), 5.14 (d, 1H, J1',2' = 8.1 Hz, H-1'), 4.63, 4.59
(2d, 2H, Jgem = 12.2 Hz, CH2Ph), 4.59-4.50 (m, 3H, H-6’a, H-6’b y CH2Ph), 4.38 (ddd, 1H,
J4',5' = 0.7 Hz, J5',6’a = 5.9 Hz, J5',6’b = 6.8 Hz, H-5'), 4.23 (d, 1H, J1,2 = 7.9 Hz, H-1), 4.19 (d,
1H, Jgem = 11.5 Hz, CH2Ph), 3.88 (dd, 1H, J5,6a = 1.8 Hz, Jgem = 10.9 Hz, H-6a), 3.71-3.65
(m, 2H, H-3 y H-6b), 5.98 (t, 1H, J3,4 = 9.3 Hz, J4,5 = 9.3 Hz, H-4), 3.46 (s, 3H, CH3O),
3.43-3.36 (m, 1H, H-5), 3.29 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 8.9 Hz, H-2) ppm. 13C NMR (126
MHz, CDCl3): δ 104.5 (C-1), 101.9 (C-1'), 86.0 (C-3), 80.6 (C-2), 75.2 (C-5), 74.0
(CH2Ph), 73.5 (CH2Ph), 71.7 (C-5'), 71.5 (C-3'), 69.7 (C-2'), 69.68 (C-6), 68.7 (C-4), 68.0
(C-4'), 62.0 (C-6'), 57.0 (CH3O) ppm.
The minor product was identified as 7β. 1H NMR (500 MHz, CDCl3): δ 5.95 dd, 1H,
J4',5' = 0.7 Hz, J3',4' = 3.4 Hz, H-4'), 5.81 (dd, 1H, J1',2' = 8.1 Hz, J2',3' = 10.4 Hz, H-2'), 5.53
14

(dd, 1H, J3',4' = 3.4 Hz, J2',3' = 10.4 Hz, H-3'), 4.85 (d, 1H, J1',2' = 8.1 Hz, H-1'), 4.80, 4.77
(2d, 2H, Jgem = 11.2 Hz, CH2Ph), 4.64-4.59 (m, 1H, H-6'a), 4.51 (dd, 1H, J5',6’b = 7.9 Hz,
Jgem = 11.7 Hz, H-6’b), 4.35-4.31 (m, 1H, H-5'), 4.33 (d, 1H, Jgem = 12.3 Hz, CH2Ph), 4.24
(d, 1H, J1,2 = 8.0 Hz, H-1), 4.16 (d, 1H, Jgem = 12.3 Hz, CH2Ph), 3.84 (dd, 1H, J2,3 = 8.7 Hz,
J3,4 = 8.7 Hz, H-3), 4.74-4.69 (m, 1H, H-4), 3.49 (s, 3H, CH3O), 4.74-4.69 (m, 1H, H-4),
3.46 (s, 1H, H-6a y H-6b), 4.43-4.36 (m, 1H, H-5), 3.29 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 8.7
13
Hz, H-2) ppm. C NMR (126 MHz, CDCl3): δ 103.9 (C-1), 101.7 (C-1'), 81.5 (C-4), 81.4
(C-2), 75.0 (C-3), 74.8 (CH2Ph), 73.4 (C-5), 73.0 (CH2Ph), 72.0 (C-5'), 71.4 (C-3'), 69.64
(C-2'), 67.99 (C-4'), 67.94 (C-6), 62.4 (C-6'), 57.0 (CH3O) ppm.
4.2.4. Methyl 2,3,4,6-tetra-O-benzoyl-
-glucopyranoside (8 ) and methyl 2,3,4,6-tetra-O-benzoyl-
(1→4)-2,6-di-O-benzoyl-a- -glucopyranoside (9
β
-
D
-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-a-
D
α
β-D
-galactopyranosyl-
D
α)
Obtained according to the general procedure by condensation of 3 with 2α. The crude
mixture showed by TLC, among the excess of 2α (Rf 0.05, 85:15 toluene/EtOAc), the
presence of a major product of Rf 0.40 and a minor product of Rf 0.37. According to the
integration of the 1H NMR signals corresponding to the OCH3 groups (3.31 and 3.34 ppm)
of both products they were in a 2.1:1.0 ratio.
Column chromatography purification (92:8 toluene-EtOAc) afforded fractions
enriched in each regioisomer. The first fractions afforded syrupy compound 8
α (0.05 g,
49%), 1H NMR (500 MHz, CDCl3): δ 8.16-7.13 (m, 30H, aromatic), 5.98 (dd, 1H, J4',5'
=
0.9 Hz, J3',4' = 3.5 Hz, H-4'), 5.82 (dd, 1H, J1',2' = 8.0 Hz, J2',3' = 10.5 Hz, H-2'), 5.58 (dd,
1H, J3',4' = 3.4 Hz, J2',3' = 10.5 Hz, H-3'), 5.04 (d, 1H, J1',2' = 8.0 Hz, H-1'), 5.02 (dd, 1H,
J1,2 = 3.8 Hz, J2,3 = 9.6 Hz, H-2), 4.98 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.69 (dd, 1H, J5,6a = 1.0
Hz, Jgem = 12.0 Hz, H-6a), 4.64-4.54 (m, 3H, H-6b, H-6’a and H-6’b), 4.47 (ddd, 1H, J4',5'
= 0.9 Hz, J5',6a' = 5.5 Hz, J5',6b' = 7.4 Hz, H-5'), 4.20 (dd, 1H, J3,4 = 8.6 Hz, J2,3 = 9.6 Hz,
13
H-3), 3.90-3.88 (m, 2H, H-4 and H-5), 3.31 (s, 3H, CH3O) ppm. C NMR (126 MHz,
CDCl3): δ 166.3, 166.1, 165.48, 165.45, 165.14, 165.10 (COPh), 133.7, 133.37, 133.34,
133.2, 133.0, 132.7, 130.0, 129.97, 129.94, 129.7, 129.6, 129.2, 129.00, 128.69, 128.67,
128.4, 128.38, 127.35, 128.31, 128.2, 128.1, 127.9 (aromatic), 102.0 (C-1'), 96.6 (C-1),
15

82.0 (C-3), 72.3 (C-2), 71.9 (C-5'), 71.4 (C-3'), 69.38 (C-2'), 69.31 (C-4), 68.6 (C-5), 67.8
(C-4'), 63.4 (C-6), 61.9 (C-6'), 55.1 (CH3O) ppm.
Further elution from the column afforded a fraction enriched in 9α (0.02 mg, 26%).
1H NMR (500 MHz, CDCl3) signals for 9α: δ 8.14-7.14 (m, 30H, aromatic), 5.97 (d, 1H,
J3',4' = 3.4 Hz, H-4'), 5.91 (dd, 1H, J1',2' = 8.0 Hz, J2',3' = 10.5 Hz, H-2'), 5.58 (dd, 1H, J3',4'
=
3.4 Hz, J2',3' = 10.5 Hz, H-3'), 5.08 (dd, 1H, J1,2 = 3.7 Hz, J2,3 = 10.0 Hz, H-2), 5.03 (d, 1H,
J1',2' = 8.0 Hz, H-1'), 4.92 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.67 (d, 1H, J = 7.8 Hz, H-6’a), 4.46-
4.41 (m, 3H, H-5', H-6a and H-6’b), 3.37 (ddd, 1H, J3,OH = 1.6 Hz, J3,4 = 8.4 Hz, J2,3 = 10.0
Hz, H-3), 4.19 (dd, 1H, J5,6b = 4.1 Hz, Jgem = 12.0 Hz, H-6b), 4.02 (ddd, 1H, J5,6a = 1.6 Hz,
J5,6b = 4.1 Hz, J4,5 = 9.9 Hz, H-5), 3.83 (dd, 1H, J3,4 = 8.4 Hz, J4,5 = 9.9 Hz, H-4), 3.34 (s,
13
3H, CH3O) ppm. C NMR (126 MHz, CDCl3): δ 166.1, 166.0, 165.5, 165.4, 165.3, 165.2
(COPh), 133.7, 133.5, 133.3, 133.2, 133.1, 130.0, 129.9, 129.8, 129.7, 129.68, 129.66,
129.62, 129.5, 129.2, 128.8, 128.7, 128.45, 128.40, 128.37, 127.33, 128.31, 128.2, 128.0
(aromatic), 102.2 (C-1'), 97.1 (C-1), 82.7 (C-4), 72.5 (C-2), 72.4 (C-5'), 71.5 (C-3'), 69.9
(C-3), 69.4 (C-2'), 67.9 (C-4'), 67.3 (C-5), 62.7 (C-6'), 62.3 (C-6), 55.4 (CH3O) ppm.
4.2.5. Methyl 2,3,4,6-tetra-O-benzoyl-
-galactopyranoside (8 ) and methyl 2,3,4,6-tetra-O-benzoyl-
(1→4)-2,6-di-O-benzoyl- -glucopyranoside (9
β
-
D
-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-
β-
D
β
β-D
-galactopyranosyl-
β
-D
β)
Obtained according to the general procedure by condensation of 3 with 2β. TLC
analysis of the crude reaction showed total consumption of 3 and a single spot of Rf 0.57
1
(85:15 toluene-EtOAc) composed, according to the H NMR spectrum of the crude
mixture, by two regioisomers in 1:5.6 ratio. The products could not be separated by column
chromatography (0.08 g; 90%) but all the NMR signals were assigned.
The minor product was identified as 8β. 1H NMR (500 MHz, CDCl3): δ 5.94 (d, 1H,
J3',4' = 3.5 Hz, H-4'), 5.82 (dd, 1H, J1',2' = 8.0 Hz, J2',3' = 10.5 Hz, H-2'), 5.51 (dd, 1H, J3',4'
=
3.5 Hz, J2',3' = 10.5 Hz, H-3'), 5.28-5.22 (m, 1H, H-2), 4.93 (d, 1H, J1',2' = 8.0 Hz, H-1'),
4.77 (dd, 1H, J5,6a = 4.8 Hz, Jgem = 11.5 Hz, H-6a), 4.66 (dd, 1H, J5',6’a = 2.0 Hz, Jgem = 11.8
Hz, H-6’a), 4.57-4.48 (m, 2H, H-6b y H-6’b), 4.47-4.39 (m, 2H, H-1 y H-5’), 4.25 (d, 1H,
J4,OH = 0.8 Hz, OH-4), 3.96 (ddd, 1H, J4,OH = 0.8 Hz, J3,4 = 8.3 Hz, J4,5 = 9.6 Hz, H-4),
3.92-4.87 (m, 1H, H-3), 3.63 (ddd, 1H, J5,6b = 2.0 Hz, J5,6a = 4.8 Hz, J4,5 = 9.6 Hz, H-5),
16

13
3.36 (s, 3H, CH3O) ppm. C NMR (126 MHz, CDCl3): δ102.1 (C-1'); 102.0 (C-1); 85.9
(C-3); 73.8 (C-5); 72.0 (C-2); 71.9 (C-5'); 71.3 (C-3'); 69.3 (C-2'); 68.8 (C-4); 67.8 (C-4');
63.4 (C-6); 62.2 (C-6'); 56.5 (CH3O) ppm.
The major product was identified as 9β. 1H NMR (500 MHz, CDCl3): δ 8.13-7.01
(m, 30H, aromatic), 5.96 (d, 1H, J3',4' = 3.4 Hz, H-4'), 5.90 (dd, 1H, J1',2' = 8.0 Hz, J2',3'
=
10.4 Hz, H-2'), 5.58 (dd, 1H, J3',4' = 3.4 Hz, J2',3' = 10.4 Hz, H-3'), 5.23 (dd, 1H, J1,2 = 8.0
Hz, J2,3 = 9.5 Hz, H-2), 5.03 (d, 1H, J1',2' = 8.0 Hz, H-1'), 4.68 (dd, 1H, J5',6a' = 1.1 Hz, Jgem
= 9.6 Hz, H-6’a), 4.54 (d, 1H, J3,OH = 1.8 Hz, OH-3), 4.51 (dd, 1H, J5,6a = 1.6 Hz, Jgem
=
12.0 Hz, H-6a), 4.50 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.43-4.36 (m, 2H, H-5' and H-6’b), 4.16
(dd, 1H, J5,6b = 4.2 Hz, Jgem = 12.0 Hz, H-6b), 4.09 (ddd, 1H, J3,OH = 1.8 Hz, J3,4 = 8.5 Hz,
J2,3 = 9.5 Hz, H-3), 3.91 (dd, 1H, J3,4 = 8.5 Hz, J4,5 = 9.8 Hz, H-4), 3.75 (ddd, 1H, J5,6a
=
1.6 Hz, J5,6b = 4.2 Hz, J4,5 = 9.8 Hz, H-5), 3.40 (s, 3H, CH3O) ppm. 13C NMR (126 MHz,
CDCl3): δ 166.1, 165.4, 165.37, 165.30, 165.29, 165.25 (COPh), 133.7, 133.5, 133.3,
133.2, 133.0, 132.9, 129.99, 129.97, 129.8, 129.76, 129.70, 129.6, 129.59, 129.55, 129.4,
129.0, 128.7, 128.67, 128.64, 128.42, 128.40, 128.38, 128.34, 128.28, 128.23, 128.20,
128.19, 128.15, 128.0 (aromatic), 102.3 (C-1'), 101.6 (C-1), 82.3 (C-4), 73.5 (C-3), 72.7
(C-2), 72.5 (C-5'), 72.0 (C-5), 71.4 (C-3'), 69.4 (C-2'), 67.9 (C-4'), 62.7 (C-6'), 62.2 (C-6),
56.7 (CH3O) ppm.
4.2.6. Methyl 2,3,5,6-tetra-O-benzoyl-
glucopyranoside (10 ) and methyl 2,3,5,6-tetra-O-benzoyl-
2,6-di-O-benzyl-a- -glucopyranoside (11
β
-
D
-galactofuranosyl-(1→3)-2,6-di-O-benzyl-a-
D-
α
β-D
-galactofuranosyl-(1→4)-
D
α)
Obtained according to the general procedure by condensation of 4 with 1α. TLC of
the crude mixture showed the formation of two regioisomers of Rf 0.46 and 0.50 (85:15
toluene/EtOAc), which according to the integration of the 1H NMR signals were in a 3.45:1
ratio. After purification by column chromatography (92:8 toluene/EtOAc) fractions of Rf
0.50 afforded compound 11α (0.02 g; 20%) impurified with 2,3,4,6-O-Bz-α,β-Galp. From
the mixture, signals corresponding to 11α: 1H NMR (500 MHz, CDCl3): δ 6.01 (dt, 1H,
J4',5' = 4.0 Hz, J5',6’a = 4.0 Hz, J5',6’b = 6.7 Hz, H-5'), 5.66-5.63 (m, 1H, H-3'), 5.36 (d, 1H,
J2',3' = 1.4 Hz, H-2'), 5.31 (s, 1H, H-1'), 5.00 (dd, 1H, J4',5' = 4.0 Hz, J3',4' = 5.4 Hz, H-4'),
5.66-5.63 (m, 3H, H-6'a, H-6'b y CH2Ph), 4.64 (d, 1H, Jgem = 12.1 Hz, CH2Ph), 4.62 (d, 1H,
17

J1,2 = 3.6 Hz, H-1), 4.56, 4.51 (2d, 2H, Jgem = 12.0 Hz, CH2Ph), 4.03 (ddd, 1H, J3,OH = 2.4
Hz, J3,4 = 8.5 Hz, J2,3 = 9.5 Hz, H-3), 3.87 (dd, 1H, J5,6a = 3.0 Hz, Jgem = 10.8 Hz, H-6a),
3.83 (dd, 1H, J3,4 = 8.5 Hz, J4,5 = 9.9 Hz, H-4), 3.81-3.77 (m, 1H, H-5), 3.70 (dd, 1H, J5,6b
=
1.7 Hz, Jgem = 10.8 Hz, H-6b), 3.42 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.5 Hz, H-2), 3.34 (s, 3H,
CH3O), 3.09 (d, 1H, J3,OH = 2.4 Hz, OH-3) ppm. 13C NMR (126 MHz, CDCl3): δ 106.1 (C-1'),
101.0 (C-1), 82.3 (C-2'), 81.2 (C-4'), 79.1 (C-2), 77.2 (C-3' y C-4), 73.31 (CH2Ph), 73.30
(CH2Ph), 71.9 (C-3), 70.4 (C-5'), 69.2 (C-5), 68.0 (C-6), 63.4 (C-6'), 55.2 (CH3O) ppm.
Fractions of Rf 0.46 afforded syrupy compound 10α (0.07 g, 69%), [α]D +1 (c 1,
1
CHCl3). H NMR (500 MHz, CDCl3): δ 8.09-7.13 (m, 30H, aromatic), 5.98 (ddd, 1H, J4',5'
= 4.2 Hz, J5',6a' = 4.4 Hz, J5',6b' = 6.4 Hz, H-5'), 5.67 (s, 1H, H-1'), 5.66-5.63 (m, 2H, H-2'
and H-3'), 4.89 (dd, 1H, J4',5' = 4.2 Hz, J3',4' = 4.6 Hz, H-4'), 4.77 (dd, 1H, J5',6a' = 4.4 Hz,
Jgem = 12.0 Hz, H-6'a), 4.76 (d, 1H, Jgem = 12.4 Hz, CH2Ph), 4.71 (dd, 1H, J5',6b' = 6.4 Hz,
Jgem = 12.0 Hz, H-6'b), 4.62 (d, 1H, Jgem = 12.4 Hz, CH2Ph), 4.60 (d, 1H, J1,2 = 3.6 Hz, H-
1), 4.58 (d, 1H, Jgem = 12.3 Hz, CH2Ph), 4.55 (d, 1H, Jgem = 12.3 Hz, CH2Ph), 3.98 (dd, 1H,
J3,4 = 8.8 Hz, J2,3 = 9.6 Hz, H-3), 3.73-3.64 (m, 3H, H-5, H-6a and H-6b), 3.60 (ddd, 1H,
J4,OH = 3.1, J3,4 = 8.8 Hz, J4,5 = 9.5 Hz, H-4), 3.54 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.6 Hz, H-
13
2), 3.36 (s, 3H, CH3O), 3.21 (d, 1H, J4,OH = 3.1 Hz, OH-4) ppm. C NMR (126 MHz,
CDCl3): δ 166.1, 165.6, 165.5, 165.1 (COPh), 138.0, 137.9, 133.5, 133.3, 133.2, 133.0,
129.7, 129.5, 129.3, 128.99, 128.96, 128.8, 128.4, 128.36, 128.33, 128.32, 128.2, 128.1,
128.8, 127.8, 127.5 (aromatic), 107.3 (C-1'), 98.0 (C-1), 81.63 (C-4'), 81.60 (C-2'), 81.3 (C-
3), 78.2 (C-2), 77.6 (C-3'), 73.5 (CH2Ph), 73.4 (CH2Ph), 70.39 (C-5'), 70.30 (C-5), 69.5 (C-
4), 69.2 (C-6), 63.2 (C-6'), 55.1 (CH3O) ppm. ESIMS: m/z calcd for C55H52NaO15 [M+Na]+
975.3198. Found: 975.3206
4.2.7. Methyl 2,3,5,6-tetra-O-benzoyl-
glucopyranoside (10 ) and methyl 2,3,5,6-tetra-O-benzoyl-
2,6-di-O-benzyl- -glucopyranoside (11
β
-
D
-galactofuranosyl-(1→3)-2,6-di-O-benzyl-
β-D-
β
β-D
-galactofuranosyl-(1→4)-
β
-D
β)
Obtained according to the general procedure by condensation of 4 with 1β. TLC of
the crude mixture showed the formation of two regioisomers of Rf 0.58 and 0.50 (85:15
1
toluene/EtOAc), which according to the integration of the H NMR anomeric signals were
in a 1.75:1 ratio. After purification by column chromatography (9:1 toluene/EtOAc)
18

fractions of Rf 0.58 (85:15 toluene/EtOAc) afforded 11β (0.03 g, 32%), [α]D -25 (c 1,
1
CHCl3). H NMR(500 MHz, CDCl3): δ 8.11-7.11 (m, 30H, aromatic), 6.00 (apparent dt,
1H, J4’,5’ = 3.8 Hz, J5’,6a’ = 4.1 Hz, J5’,6b’ = 7.0 Hz, H-5’), 5.62 (d, 1H, J3’,4’ = 5.4 Hz, H-3’),
5.39-5.38 (m, 2H, H-1’ and H-2’), 4.97 (dd, 1H, J4’,5’ = 3.8 Hz, J3’,4’ = 5.4 Hz, H-4’), 4.90
(d, 1H, Jgem = 11.3 Hz, CH2Ph), 4.78 (dd, 1H, J5’,6a’ = 4.1 Hz, Jgem = 12.1 Hz, H-6’a), 4.71
(dd, 1H, J5’,6b’ = 7.0 Hz, Jgem = 12.1 Hz, H-6’b), 4.69 (d, 1H, Jgem = 11.3 Hz, CH2Ph), 4.59,
4.55 (2d, 2H, Jgem = 12.1 Hz, CH2Ph), 4.31 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.86 (dd, 1H, J5,6a
=
3.9 Hz, Jgem = 11.0 Hz, H-6ª), 3.83 (apparent t, 1H, J3,4 = 9.0 Hz, J4,5 = 9.8 Hz, H-4), 3.80
(dd, 1H, J5,6b = 1.8 Hz, Jgem = 11.0 Hz, H-6b), 3.70 (td, 1H, J3,4 = 9.0 Hz, J2,3 = 9.1 Hz,
J3,OH = 1.7 Hz, H-3), 3.56 (s, 3H, CH3O), 3.50 (ddd, 1H, J5,6b = 1.8 Hz, J5,6a = 3.9 Hz, J4,5
=
9.8 Hz, H-5), 3.29 (dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.1 Hz, H-2), 3.09 (d, 1H, J3,OH = 1.7 Hz,
OH-3) ppm.13C NMR (126 MHz, CDCl3): δ 166.1, 165.7, 165.6, 165.5 (COPh), 138.4,
138.1, 133.5, 133.4, 133.3, 133.0, 130.0, 129.88, 129.84, 129.7, 129.5, 129.4, 128.9, 128.7,
128.6, 128.46, 128.41, 128.40, 128.3, 128.1, 128.0, 127.7, 127.5, 127.4 (aromatic), 106.3
(C-1’), 104.2 (C-1), 82.2 (C-2’), 81.5 (C-2), 81.2 (C-4’), 77.3 (C-3’), 77.1 (C-4), 74.9 (C-
3), 74.5 (CH2Ph), 74.4 (C-5), 73.2 (CH2Ph), 68.3 (C-5’), 68.3 (C-6), 63.5 (C-6’), 57.0
(CH3O) ppm. ESIMS: m/z calcd for C55H52NaO15 [M+H]+ 975.3198. Found: 975.3201.
1
Fractions of Rf 0.50 afforded compound 10β (0.05 g, 55%), [α]D -8 (c 1, CHCl3). H
NMR(500 MHz, CDCl3): δ 8.13-7.05 (m, 30H, aromatic), 5.98 (apparent dt ddd, 1H, J4’,5’
=
4.8 Hz, J5’,6a’ = 4.6 Hz, J5’,6b’ = 6.5 Hz, H-5’), 5.63 (s, 1H, H-1’), 5.60 (dd, 1H, J2’,3’ = 1.4
Hz, J3’,4’ = 5.1 Hz, H-3’), 5.56 (d, 1H, J2’,3’ = 1.4 Hz, H-2’), 4.87 (dd, 1H, J4’,5’ = 4.0 Hz,
J3’,4’ = 5.1 Hz, H-4’), 4.83 (d, 1H, Jgem = 10.9 Hz, CH2Ph), 4.74 (dd, 1H, J5’,6a’ = 4.6 Hz,
Jgem = 11.9 Hz, H-6’a), 4.72 (d, 1H, Jgem = 10.9 Hz, CH2Ph), 4.70 (dd, 1H, J5’,6b’ = 6.5 Hz,
Jgem = 11.9 Hz, H-6’b), 4.60, 4.57 (2d, 2H, Jgem = 12.3 Hz, CH2Ph), 4.30 (d, 1H, J1,2 = 7.9
Hz, H-1), 3.77 (dd, 1H, J5,6a = 3.3 Hz, Jgem = 10.6 Hz, H-6ª), 3.67 (apparent t, 1H, J3,4 = 9.0
Hz, J2,3 = 9.1 Hz, H-3), 3.66 (dd, 1H, J5,6b = 5.3 Hz, Jgem = 10.6 Hz, H-6b), 3.57 (s, 3H,
CH3O), 3.54 (td, 1H, J4,OH = 2.8 Hz, J3,4 = 9.0 Hz, J4,5 = 9.5 Hz, H-4), 3.43 (ddd, 1H, J5,6a
=
3.3 Hz, J5,6b = 5.3 Hz, J4,5 = 9.5 Hz, H-5), 3.42 (dd, 1H, J1,2 = 7.9 Hz, J2,3 = 9.1 Hz, H-2),
13
3.28 (d, 1H, J4,OH = 2.8 Hz, OH-4) ppm. C NMR (126 MHz, CDCl3): δ 166.1, 165.6,
165.5, 165.1 (COPh), 138.1, 138.0, 133.5, 133.3, 133.2, 133.0, 129.95, 129.92, 129.8,
129.7, 129.5, 129.3, 128.9, 128.8, 128.4, 128.39, 128.38, 128.35, 128.31, 128.26, 128.25,
19

128.1, 127.66, 127.64, 127.5 (aromatic), 106.9 (C-1’), 104.5 (C-1), 82.9 (C-3), 81.69 (C-
2’), 81.66 (C-4’), 80.6 (C-2), 77,5 (C-3’), 74.58 (CH2Ph), 74.54 (C-5), 73.5 (CH2Ph), 70.3
(C-5’), 70.2 (C-4), 69.9 (C-6), 63.2 (C-6’), 57.1 (CH3O) ppm. ESIMS: m/z calcd for
C55H52NaO15 [M+H]+ 975.3198. Found: 975.3168.
4.2.8. Methyl 2,3,5,6-tetra-O-benzoyl-
glucopyranoside (12 ) and methyl 2,3,5,6-tetra-O-benzoyl-
2,6-di-O-benzoyl-a- -glucopyranoside (13
β
-
D
-galactofuranosyl-(1→3)-2,6-di-O-benzoyl-a-
D-
α
β-D
-galactofuranosyl-(1→4)-
D
α)
Obtained according to the general procedure by condensation of 4 with 2α. After 2h
of reaction TLC analysis showed consumption of compound 4 and the formation of
products of Rf 0.28 and 0.32 (85:15 Toluene-EtOAc). According to the integration of the
1H NMR signals corresponding to H-4, they were in 2.0:1.0 ratio.
After purification by column chromatography (9:1 Toluene-EtOAc) fractions of Rf
0.32 afforded 12α (0.04 g; 38%), impurified with 13α, 1H NMR (500 MHz, CDCl3):
δ 8.12-7.21 (m, 30H, aromatic), 5.98 (dt, 1H, J4',5' = 4.1 Hz, J5',6a' = 4.6 Hz, J5',6b' = 6.6 Hz,
H-5'), 5.68 (dd, 1H, J2',3' = 1.8 Hz, J3',4' = 5.5 Hz, H-3'), 5.49 (dd, 1H, J1',2' = 0.7 Hz, J2',3'
=
1.8 Hz, H-2'), 5.48 (s, 1H, H-1'), 5.07-5.02 (m, 2H, H-1 and H-2), 4.94 (dd, 1H, J4',5' = 4.1
Hz, J3',4' = 5.5 Hz, H-4'), 4.79 (dd, 1H, J5',6a' = 4.6 Hz, Jgem = 12.0 Hz, H-6’a), 4.78 (dd,
1H, J5,6a = 1.9 Hz, Jgem = 11.9 Hz, H-6a), 4.69 (dd, 1H, J5,6b = 4.4 Hz, Jgem = 11.9 Hz, H-
6b), 4.68 (dd, 1H, J5',6b' = 6.6 Hz, Jgem = 12.0 Hz, H-6’b), 4.31 (td, 1H, J3,OH = 3.2 Hz, J3,4
= 8.8 Hz, J2,3 = 8.9 Hz, H-3), 4.14 (ddd, 1H, J5,6a = 1.9 Hz, J5,6b = 4.4 Hz, J4,5 = 10.0 Hz,
H-5), 3.88 (dd, 1H, J3,4 = 8.8 Hz, J4,5 = 10.0 Hz, H-4), 3.64 (d, 1H, J3,OH = 3.2 Hz, OH-3),
3.42 (s, 3H, CH3O) ppm. 13C NMR (126 MHz, CDCl3): δ 166.19, 166.12, 165.65, 165.61,
163.5 (COPh), 133.6, 133.4, 133.3, 133.2, 133.1, 133.0, 129.9, 129.88, 129.85, 129.7,
129.6, 129.3, 129.1, 128.9, 128.7, 128.6, 128.49, 128.40, 128.35, 128.34 (aromatic), 107.5
(C-1'), 97.1 (C-1), 82.7 (C-2'), 81.6 (C-4'), 79.8 (C-4), 77.1 (C-3'), 73.4 (C-2), 70.3 (C-3),
70.3 (C-5'), 67.9 (C-5), 63.0 (C-6), 62.9 (C-6'), 55.5 (CH3O) ppm.
Fractions of Rf 0.28 afforded a sample enriches in 13α (0.05 g; 54%). 1H NMR (500
MHz, CDCl3): 8.10-7.13 (m, 30H, aromatic), 5.96 (ddd, 1H, J4',5' = 4.3 Hz, J5',6a' = 4.7 Hz,
J5',6b' = 6.3 Hz, H-5'), 5.62 (dd, 1H, J2',3' = 1.9 Hz, J3',4' = 5.5 Hz, H-3'), 5.55 (s, 1H, H-1'),
5.42 (dd, 1H, J1',2' = 0.8 Hz, J2',3' = 1.9 Hz, H-2'), 5.14 (dd, 1H, J1,2 = 3.7 Hz, J2,3 = 10.0
20

Hz, H-2), 5.07 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.9 (dd, 1H, J4',5' = 4.3 Hz, J3',4' = 5.5 Hz, H-4'),
4.79 (dd, 1H, J5',6a' = 4.7 Hz, Jgem = 11.9 Hz, H-6’a), 4.72 (dd, 1H, J5',6b' = 6.3 Hz, Jgem
11.9 Hz, H-6’b), 4.65-4.57 (m, 2H, H-6a and H-6b), 4.25 (dd, 1H, J3,4 = 9.0 Hz, J2,3
=
=
10.0 Hz, H-3), 3.98 (ddd, 1H, J5,6a = 2.9 Hz, J5,6b = 4.6 Hz, J4,5 = 9.9 Hz, H-5), 3.71 (dd,
1H, J3,4 = 9.0 Hz, J4,5 = 9.9 Hz, H-4), 3.64 (br. s, 1H, OH-4), 3.39 (s, 3H, CH3O) ppm. 13C
NMR (126 MHz, CDCl3): δ 166.6, 166.2, 165.8, 165.6, 165.5, 164.8 (COPh), 133.5,
133.3, 133.2, 133.17, 133.13, 133.0, 129.96, 129.92, 129.88, 129.82, 129.7, 129.46,
129.40, 129.3, 129.0, 128.7, 128.5, 128.4, 128.3, 128.27, 128.23, 128.20, (aromatic),
107.4 (C-1'), 97.0 (C-1), 81.9 (C-2'), 81.6 (C-4'), 79.7 (C-3), 77.05 (C-3'), 72.7 (C-2), 70.3
(C-5'), 69.7 (C-3), 69.5 (C-4), 63.6 (C-6), 63.0 (C-6'), 55.2 (CH3O) ppm.
4.2.9. Methyl 2,3,5,6-tetra-O-benzoyl-
-glucopyranoside (12 ) and methyl 2,3,5,6-tetra-O-benzoyl-
(1→4)-2,6-di-O-benzoyl- -glucopyranoside (13
β
-
D
-galactofuranosyl-(1→3)-2,6-di-O-benzoyl-
β-
D
β
β-D
-galactofuranosyl-
β
-D
β)
Obtained according to the general procedure by condensation of 4 with 2β. 1H NMR
spectrum of the crude mixture evidenced the formation of two disaccharides in a 1:1 ratio,
according to the integration of the H-3’ signals. After purification by column
chromatography (9:1 toluene-hexane) fractions of Rf 0.47 (85:15 toluene-hexane)
1
afforded compound 13β (0.04 g, 42%), [α]D -46 (c 1, CHCl3). H NMR (500 MHz,
CDCl3): 1H NMR (500 MHz, CDCl3): δ 8.11-7.12 (m, 30H, aromatic), 5.95 (ddd, 1H, J4',5'
= 4.1 Hz, J5',6a' = 4.6 Hz, J5',6b' = 6.5 Hz, H-5'), 5.66 (dd, 1H, J2',3' = 1.8 Hz, J3',4' = 5.5 Hz,
H-3'), 5.47 (dd, 1H, J1',2' = 0.6 Hz, J2',3' = 1.8 Hz, H-2'), 5.45 (s, 1H, H-1'), 5.18 (dd, 1H,
J1,2 = 8.0 Hz, J2,3 = 9.1 Hz, H-2), 4.92 (dd, 1H, J4',5' = 4.1 Hz, J3',4' = 5.5 Hz, H-4'), 4.83
(dd, 1H, J5,6a = 1.9 Hz, Jgem = 12.1 Hz, H-6a), 4.76 (dd, 1H, J5',6a' = 4.6 Hz, Jgem = 12.0 Hz,
H-6’a), 4.66 (dd, 1H, J5',6b' = 6.5 Hz, Jgem = 12.0 Hz, H-6’b), 4.65 (dd, 1H, J5,6b = 4.4 Hz,
Jgem = 12.1 Hz, H-6b), 4.57 (d, 1H, J1,2 = 8.0 Hz, H-1), 3.99 (ddd, 1H, J3,OH = 3.6 Hz, J3,4
= 8.7 Hz, J2,3 = 9.1 Hz, H-3), 3.94 (apparent t, 1H, J3,4 = 8.7 Hz, J4,5 = 9.3 Hz, H-4), 3.85
(ddd, 1H, J5,6a = 1.9 Hz, J5,6b = 4.4 Hz, J4,5 = 9.3 Hz, H-5), 3.75 (d, 1H, J3,OH = 3.6 Hz,
13
OH-3), 3.48 (s, 3H, CH3O) ppm. C NMR (126 MHz, CDCl3): δ 166.11, 166.10, 165.7,
165.6, 165.58, 165.54 (COPh), 133.5, 133.39, 133.30, 133.15, 133.11, 132.9, 129.95,
129.93, 129.91, 129.86, 129.83, 129.7, 129.6, 129.3, 129.1, 129.0, 128.7, 128.46, 128.44,
21

128.38, 128.36, 128.33, 128.31, 128.30, 128.2 (aromatic), 107.5 (C-1'), 101.7 (C-1), 82.6
(C-2'), 81.6 (C-4'), 79.5 (C-4), 77.1 (C-3'), 73.9 (C-3), 73.8 (C-2), 72.6 (C-5), 70.3 (C-5'),
2 x 68.3 (C-6 and C-6'), 56.8 (CH3O) ppm. ESIMS: m/z calcd for C55H48NaO17 [M+H]+
1003.2784. Found: 1003.2771.
Fractions of Rf 0.43 (85:15 toluene-hexane) afforded compound 12β 0.05 g, 47%),
1
[α]D +8 (c 1, CHCl3). H NMR (500 MHz, CDCl3): δ 8.11-7.13 (m, 30H, aromatic), 5.93
(ddd, 1H, J4',5' = 4.3 Hz, J5',6a' = 4.8 Hz, J5',6b' = 6.2 Hz, H-5'), 5.59 (dd, 1H, J2',3' = 1.9 Hz,
J3',4' = 5.4 Hz, H-3'), 5.43 (s, 1H, H-1'), 5.35 (dd, 1H, J1',2' = 0.8 Hz, J2',3' = 1.9 Hz, H-2'),
5.32 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.5 Hz, H-2), 4.86 (dd, 1H, J4',5' = 4.3 Hz, J3',4' = 5.4 Hz,
H-4'), 4.78 (dd, 1H, J5',6a' = 4.8 Hz, Jgem = 11.9 Hz, H-6’a), 4.67 (dd, 1H, J5',6b' = 6.2 Hz,
Jgem = 11.9 Hz, H-6’b), 4.64 (dd, 1H, J5,6a = 2.2 Hz, Jgem = 12.0 Hz, H-6a), 4.57 (dd, 1H,
J5,6b = 5.1 Hz, Jgem = 12.0 Hz, H-6b), 4.50 (d, 1H, J1,2 = 8.0 Hz, H-1), 3.91 (dd, 1H, J3,4
=
8.7 Hz, J2,3 = 9.6 Hz, H-3), 3.73 (ddd, 1H, J4,OH = 3.3 Hz, J3,4 = 8.7 Hz, J4,5 = 9.6 Hz, H-4),
3.67 (ddd, 1H, J5,6a = 2.2 Hz, J5,6b = 5.1 Hz, J4,5 = 9.6 Hz, H-5), 3.66 (d, 1H, J3,OH = 3.3 Hz,
13
OH-4), 3.48 (s, 3H, CH3O) ppm. C NMR (126 MHz, CDCl3): δ 166.6, 166.1, 165.59,
165.54, 165.1, 164.7 (COPh), 133.5, 133.3, 133.2, 133.1, 132.8, 132.9, 129.99, 129.92,
129.8, 129.78, 129.74, 129.6, 129.4, 129.2, 129.0, 128.9, 128.7, 128.59, 128.51, 128.45,
128.41, 128.3, 128.2, 128.1 (aromatic), 107.4 (C-1'), 101.9 (C-1), 83.0 (C-3), 82.0 (C-2'),
81.7 (C-4'), 76.8 (C-3'), 74.0 (C-5), 72.4 (C-2), 70.3 (C-5'), 69.5 (C-4), 63.6 (C-6), 62.9 (C-
6'), 56.8 (CH3O) ppm. ESIMS: m/z calcd for C55H48NaO17 [M+H]+ 1003.2784. Found:
1003.2808.
4.3. Computational methods
Molecular mechanics calculations were carried out using the program MM3(92)
(QCPE, Indiana, USA)51,52 Quantum mechanical calculations were carried out using
Gaussian 09W (rev. C.01),53 with standard termination options.
From a given rotamer, an automated routine was used to generate the starting
conformations produced by rotation of ± 120º for each of the exocyclic dihedrals. Those
conformers within the first 10 kcal/mol were submitted to DFT optimizations at the
B3LYP/6-311+G** level and then to single point calculations with M06-2X at the same
level. Charges were then obtained with M06-2X at the same level of theory, for both the
22

ground molecule and the radical cation, using the Mulliken charge distribution. Condensed-
to-atom Fukui functions and charges were then obtained as Boltzmann average ratios
Stationary points were characterized by frequency calculations in order to verify that
the minima had no imaginary frequencies. Population analysis was carried out using the
Boltzmann equation, with a temperature of 298 K and using electronic energies for DFT.
Declaration of competing interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Acknowledgements
This work was supported by grants from Universidad de Buenos Aires (UBA),
Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina. CAS and
CM are Research Members of CONICET. EADV was supported by a fellowship from
CONICET.
Appendix A. Electronic Supplementary Information (ESI)
Supplementary data to this article (Additional figure and tables and NMR spectra
for compounds 6-13) can be found at https: doi:…….
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ꢀ Glucopyranosyl acceptors 2,6-di-O-protected were efficiently synthesized.
ꢀ Relative reactivity of O-3/O-4 of glucopyranosides in glycosylation reactions was
analyzed.
ꢀ The α-anomers are preferentially glycosylated at O-3. The benzoylated β-anomer is
preferentially glycosylated at O-4.
ꢀ Experimental regioselectivities and molecular modeling were compared.
ꢀ 1→3 or 1→4 Linkages can selectively be installed using the appropriate glucosyl
acceptor.