Synthesis and NMR analysis of model compounds related to fucosylated chondroitin sulfates: GalNAc and Fuc(1?→?6)GalNAc derivatives

Article abstract of DOI:10.1016/j.carres.2016.11.015

Unsubstituted and 6-O-α-L-fucosylated propyl 2-acetamido-2-deoxy-β-D-galactopyranosides and their selectively O-sulfated (both in GalNAc and Fuc units) derivatives were synthesized as model compounds representing the fragments of fucosylated chondroitin s

Full text of DOI:10.1016/j.carres.2016.11.015

Accepted Manuscript
Synthesis and NMR analysis of model compounds related to fucosylated chondroitin
sulfates: GalNAc and Fuc(1 → 6)GalNAc derivatives
Dmitry Z. Vinnitskiy, Nadezhda E. Ustyuzhanina, Andrey S. Dmitrenok, Alexander S.
Shashkov, Nikolay E. Nifantiev
PII:
S0008-6215(16)30546-8
10.1016/j.carres.2016.11.015
CAR 7298
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 16 November 2016
Revised Date: 25 November 2016
Accepted Date: 25 November 2016
Please cite this article as: D.Z. Vinnitskiy, N.E. Ustyuzhanina, A.S. Dmitrenok, A.S. Shashkov, N.E.
Nifantiev, Synthesis and NMR analysis of model compounds related to fucosylated chondroitin
sulfates: GalNAc and Fuc(1 → 6)GalNAc derivatives, Carbohydrate Research (2016), doi: 10.1016/
j.carres.2016.11.015.
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ACCEPTED MANUSCRIPT
Synthesis and NMR analysis of model compounds related to fucosylated chondroitin
sulfates: GalNAc and Fuc(1→6)GalNAc derivatives
D. Z. Vinnitskiy,a N. E. Ustyuzhanina,a A. S. Dmitrenok,a A. S. Shashkov,a and N. E. Nifantieva

ACCEPTED MANUSCRIPT
Synthesis and NMR analysis of model compounds related to fucosylated chondroitin
sulfates: GalNAc and Fuc(1→6)GalNAc derivatives
Dmitry Z. Vinnitskiy, Nadezhda E. Ustyuzhanina,* Andrey S. Dmitrenok, Alexander S.
Shashkov, and Nikolay E. Nifantiev*
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect
47, Moscow 119991, Russia
*Corresponding authors: Tel/Fax: +7-499-135-87-84; e-mail: ustnad@gmail.com, nen@ioc.ac.ru
Abstract. Unsubstituted and 6-O-α-L-fucosylated propyl 2-acetamido-2-deoxy-β-D-
galactopyranosides and their selectively O-sulfated (both in GalNAc and Fuc units) derivatives
were synthesized as model compounds representing the fragments of fucosylated chondroitin
sulfates (FCS) from sea cucumbers. Per-O-acetylated 2-deoxy-2-N-phthalimido-D-
glucopyranose was used as a key precursor for the preparation of all 2-acetamido-2-deoxy-D-
galactopyranoside containing products. Attempts at 6-O-glycosylation of propyl 3-O-benzoyl-2-
deoxy-2-N-phthalimido-D-galactoside
by
2-O-benzyl-3,4-di-O-chloracetyl-L-fucosyl
trichloracetimidate in the presence of TMSOTf gave a 1:1 mixture of the corresponding α- and
β-isomeric disaccharides, while the use of structurally related fucosyl bromide donor with
promotion by Bu4NBr led to the formation of desired α-isomeric disaccharide exclusively.
Selective removal of orthogonal O-protections permitted subsequent O-sulfation both at the
GalNAc and Fuc units. Further removal of blocking groups yielded the target products which
were systematically studied by 1H and 13C NMR spectroscopy in order to determine the spectral
effects of O-sulfation and α-L-fucosylation needed for the development of computer assisted
structural analysis of natural FCS.

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Keywords: fucosylated chondroitin sulfate; oligosaccharide; synthesis; glycosylation; NMR;
spectral glycosylation effect; spectral sulfation effect
1. Introduction
Fucosylated chondroitin sulfates (FCS) from sea cucumbers attract attention due to
their high level of anticoagulant, antithrombotic, antitumor, anti-inflammatory, and other
types of biological activity [1-4]. The backbone of FCS is built of the repeating
disaccharide units
branches (Fig. 1). Structural variations of FCS from different sea cucumber species are
determined by the number and position of branches, as well as by the pattern of
→3)-β-D-GalNAc-(1
→4)-β-D-GlcA-(1
→
bearing α-L-fucosyl
O-
sulfation of all monosaccharide units. It is noteworthy that biological effect depends on
these structural features of the polysaccharide [3-6]. For the two last decades selectively
O
-sulfated α-L-fucosyl residues attached to
considered to be the main type of branches in FCS [1, 5-8]. However, recently the
presence of α-L-fucosyl branches linked to -6 of N-acetyl-galactosamine units (see unit
O-3 of the GlcA unit (see unit A, Fig. 1) were
O
II, Fig. 1) was found in the structure of FCS from Actinopyga mauritiana [9].
Here, we conduct systematic syntheses of oligosaccharides related to essential
structural motifs of FCS. These compounds are required for subsequent biological studies
to assess the structures of pharmacophore fragments in FCS [10-11]. In addition, the same
compounds are indispensable models for NMR studies to determine spectral effects of
glycosylation and sulfation which are needed for the development of computer assisted
analysis of natural FCS [12-13].
Previously we performed the synthesis of oligosaccharides built of 3-
O-
fucosylated glucuronic acid [14]. In this communication we report the synthesis and
NMR analysis of mono- and disaccharides
fragments of FCS (Fig. 1).
1-8 related to GalNAc and Fuc(1→6)GalNAc

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Figure 1. Key repeating units
I
and II of fucosylated chondroitin sulfates and synthetic mono-
6)GalNAc fragments.
and disaccharides related to GalNAc and Fuc(1→
1
-8
2. Results and Discussion
2.1. Synthesis
The synthesis of all N-acetyl-D-galactosamine derivatives was started from per-
acetylated 2-deoxy-2-phthalimido-D-glucose [15]. Its treatment with allyl alcohol in the
presence of BF3 Et2O gave the respective allyl glycoside, which was -deacetylated
under acidic conditions and further transformed to 3- -acetyl-4,6-di- -benzylidene
O-
9
O
O
O
derivative 10 in overall yield of 64% (Scheme 1). Regioselective reductive opening of the
benzylidene ring in 10 under the treatment with BH3 Me3N and AlCl3 in THF gave 6-O-
benzylated glycoside 11 in a yield of 90%. Presence of the benzyl group at
O-6 was
confirmed by the low field shift of the
C
-6 signal in the 13C NMR spectrum (
δ 70.2 ppm).
Transformation of compound 11 with gluco-configuration into galacto-derivative 12 was
performed via intermediate formation of 4-triflate of 11. Intramolecular attack of the 3-
acetyl group on the triflate group at -4 led to the dioxalenium cation, which was opened
in the presence of water with the formation of 4- -acetylated product 12. The presence of
-4 in 12 was confirmed by the low field shift of the -4 signal in the
5.44 ppm). Galacto-configuration of 12 was evidenced by the
O-
C
O
the acetyl group at
O
H
1H NMR spectrum (
δ

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characteristic value of J3-4 constant (3.4 Hz). Deprotection of 12 accompanied by
reduction of the allyl group into the propyl one and followed by N-acetylation led to the
target monosaccharide 1.
Scheme 1. i: 1. AllOH, BF3·Et2O, DCM, 2. AcCl/MeOH, DCM 3. PhC(OMe)2, CSA, DMF, 4.
AcCl, Py, DCM, 64%; ii: BH3·NMe3, AlCl3, H2O, THF, 90%; iii: 1. Tf2O, Py, DCM, -50 °C, 2.
H2O, DCM, 50 °C, 94%; iv: 1. Pd(OH)2/C, EtOAc, MeOH, 2. N2H4·H2O, EtOH, 90 °C, 3. Ac2O,
NaHCO3, MeOH, H2O, 56%; v: BzCl, Py, DCM, 91%; vi: 1. AcCl/MeOH, DCM 2. Py·SO3,
DMF, 3. Pd(OH)2/C, EtOAc, MeOH, 4. N2H4·H2O, EtOH, 90 °C, 5. Ac2O, NaHCO3, MeOH,
H2O, 48%; vii: Pd(OH)2/C, EtOAc, MeOH, 89%; viii: AcCl/MeOH, DCM, 96%; ix: 1. Py·SO3,
DMF, 2. N2H4·H2O, EtOH, 90 °C, 3. Ac2O, NaHCO3, MeOH, H2O, 52% for 3, 48% for 4.
Benzoylation of 12 gave glycoside 13 bearing the orthogonal 4-
benzyl groups. Acidic -deacetylation of 13 followed by sulfation, deprotection and,
finally N-acetylation, led to the target 4- -sulfated monosaccharide in an overall yield
of 48%. Hydrogenolysis of 13 in the presence of Pd(OH)2/C gave propyl glycoside 14
bearing free OH-group at -6. -Sulfation of 14 and subsequent deprotection and N-
acetylation produced 6- -sulfated in a yield of 52%. Similarly, -deacylation of 14
(→15, 96%) and subsequent sulfation, saponification and N-acetylation gave 4,6-di-
sulfated propyl glycoside . The position of the sulfate groups in compounds was
O-acetyl and 6-O-
O
O
2
C
O
O
3
O
O-
4
2-4
confirmed by characteristic low field values of the corresponding proton and carbon
chemical shifts in the 1H and 13C NMR spectra (Table 2).

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For the synthesis of disaccharides the glycosylation of diol 15 by 2-O-benzyl-
5
-
8
3,4-di-
O-chloroacetyl-fucosyltrichloroacetimidate 16 [14] in the presence of TMSOTf
was studied first. Previously, 3,4-di-
O-acyl-fucosyltrichloroacetimidates were shown to
be efficient α-glycosylating agents due to remote participation of acyl groups at O-3
and/or O-4 which favour nucleophilic attack from the α-side [14, 16-18]. In spite of
previous observations, the reaction of 15 with 16 proceeded without any stereoselectivity
and led to a 1:1 mixture of α- and β-isomers 18αβ (Table 1). Their ratio was determined
by the integration of H-6(α-Fuc) and H-6(β-Fuc) signals in the 1H NMR spectrum.
Table 1. Results of glycosylation.
Donor
Acceptor
Conditions
TMSOTf, -50
°C,
Products
Yield
51%
α:β
1:1
DCM, 30 min
TMSOTf, -50
°C,
78%
2:1
DCM, 30 min
Bu4NBr, rt,
DCM, 7 days
56% only α
Fucosylation under similar conditions of 6-hydroxy derivative 14 which can be
regarded as a less reactive acceptor was slightly more efficient and led to a 2:1 mixture of
α- and β-isomers 19αβ (Table 1). Their ratio was determined by the integration of H-1(α-
1
Fuc) and H-1(β-Fuc) signals in the H NMR spectrum. Surprisingly, the best result of

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glycosylation was achieved with the use of the less active fucosyl bromide donor 17
bearing the same pattern of blocking groups as that in imidate 16. The coupling of
compounds 15 and 17 under Lemieux conditions [19] in the presence of Bu4NBr
proceeded more slowly than that with trichloroacetimidate 16 (7 days instead of 30 min,
Table 1) but with stereospecific formation of the desired α-isomer 18α. α-Configuration
of the formed glycosidic bond was confirmed by the characteristic value of the J1’-2’
constant (3.4 Hz).
Disaccharide 18α was further transformed into the target products
5-
8
. Thus, the
deprotection and N-acetylation of 18α gave non-sulfated disaccharide
5, while the
sulfation of 18α by the Py·SO3 complex followed by deprotection and N-acetylation led
to 4- -sulfated disaccharide . Selective removing of chloroacetyl groups in 18α by
thiourea in the presence of collidine gave triol 20 (92%). It was then subjected to per-
sulfation followed by further deprotection and N-acetylation to give 4,3’,4’-tri- -sulfated
disaccharide . Hydrogenolysis of 20 followed by per- -sulfation, deprotection and N-
acetylation led to 4,2’,3’,4’-tetra- -sulfated disaccharide . Similarly to monosaccharide
sulfates , the location of the sulfate groups in compounds was confirmed by the
O
6
O-
O
7
O
O
8
2
-
4
6-8
characteristic low field values of the corresponding proton and carbon chemical shifts in
the 1H and 13C NMR spectra (Table 2).
Scheme 2
MeOH, H2O, 51%. ii: 1. Py·SO3, DMF, 2. Pd(OH)2/C, EtOAc, MeOH, 3. N2H4·H2O, EtOH, 90
°C, 4. Ac2O, NaHCO3, MeOH, H2O, 42% for , 37% for iii: Thiourea, 2,4,6-collidine, MeOH,
. i: 1. Pd(OH)2/C, EtOAc, MeOH, 2. N2H4·H2O, EtOH, 90 °C, 3. Ac2O, NaHCO3,
6
7.

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60 °C, 92%. iv: 1. Pd(OH)2/C, EtOAc, MeOH, 2. Py·SO3, DMF, 3. N2H4·H2O, EtOH, 90 °C, 4.
Ac2O, NaHCO3, MeOH, H2O, 47%.
2.2. NMR analysis
1
13
All synthesized mono- and disaccharides 1-8 were characterized by H and C
NMR spectroscopy (Table 2) in order to determine the corresponding spectral effects of
sulfation and glycosylation, which could be applied to the analysis of complicated NMR
spectra of FCS which can be illustrated by the analysis of the structure of natural sulfated
polysaccharide fucoidan from Fucus distichus [13].
The effects of sulfation or glycosylation on protons (ꢀδHi) and carbons (ꢀδCi)
were calculated according to typical equations: ꢀδHi = δHi(A) – δHi(B) and ꢀδCi =
δCi(A) – δCi(B) [10, 20, 21], where A was the sulfated or glycosylated product and B
was the corresponding non-sulfated or non-glycosylated product, respectively (Table 3).
Thus, for the calculation of sulfation effects in the case of monosaccharides
galactosaminide was accounted as reference B, while for disaccharides it was
compound . It was found that the carbon α-effects of sulfation ꢀδCi were always
2-4,
1
6-8
5
positive and amounted to a value of 6.1-8.2 ppm, while the β-effects were usually
negative and did not exceed the value of -2.8 ppm. The same trend was observed
previously for oligosaccharides related to fucoidan fragments [20-22]. The proton α-
effects of sulfation ꢀδHi were also positive, and the value of ꢀδH4 was significantly
higher than that of ꢀδH6. In contrast to the carbon β-effects, the values of proton β-
effects were positive and could amount to +0.35 ppm. This could be explained by the
negative inductive effect of the sulfate group on neighbouring protons.
The influence of the sulfate group at O-2 of the fucosyl residue on the NMR
spectra was determined by comparing the chemical shifts of the respective signals of
compounds
7 and 8 (Table 3, last entry). In addition to the potent α-effect (ꢀδH-2 = +0.54
ppm, ꢀδC-2 = +6.4 ppm), significant β-effects on H-1 and C-1 atoms (ꢀδH-1 = +0.27

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ppm, ꢀδC-1 = -2.7 ppm) were determined. The later observation could be helpful for
identification of the signals of Fuc2S(1
spectra of fucosylated chondroitin sulfates.
The proton and carbon effects of 6-O-glycosylation in the spectra of disaccharides
were calculated with the use of spectral data for 4-O-sulfated galactosaminide as
→6) residue in the anomeric region of the NMR
6
-
8
2
reference B. The values ꢀδC6 were shown to be significant indicating the potent α-effect
of glycosylation comparable with the α-effect of sulfation (Table 3). This makes
impossible identification of 6-O-sulfated or 6-O-fucosylated GalNAc units only on the
basis of chemical shift values of C-6 signals in the NMR spectra of natural FCS. The β-
effects of glycosylation on 13C (negative) and 1H (positive) were also comparable with the
corresponding spectral β-effects of sulfation.
Notably, the introduction of substituents both at O-4 and O-6 of GalNAc causes a
distinction of usually equal CH2 protons of GalNAc (see the data for
4 and 6-8). Overall,
the parent NMR chemical shifts of model compounds together with the calculated
1-8
sulfation and fucosylation effects forms a useful data set which could be applied for the
analysis of NMR spectra of natural FCS.
1
Table 2. Data of H and 13C NMR spectra (
δ
, ppm) of
1
-
8
(bold numerals indicate
positions of sulfates).
Residue
H-1
(C-1)
4.49
H-2
(C-2) (C-3) (C-4) (C-5)
3.90 3.74 3.96 3.70
H-3
H-4
H-5
H-6
(C-6)
3.81
1 GalNAc
2 GalNAc
3 GalNAc
(103.0) (53.9) (72.5) (69.3) (76.5)
4.54 3.90 3.90 3.82
(103.4) (54.5) (72.0) (77.5) (76.2)
4.49 3.91 3.75 4.00 3.93
(62.4)
3.83
4.71
(62.5)
4.22

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(103.0) (53.9) (72.4) (69.0) (74.0)
4.52 3.89 3.89 4.05
(102.7) (54.1) (71.4) (77.0) (74.0)
4.49 3.90 3.72 3.96 3.88
(103.0) (53.9) (72.4) (69.5) (75.0)
4.97 3.80 3.86 3.81 4.10
(100.7) (69.3) (71.0) (73.2) (68.2)
4.56 3.90 3.90 4.00
(102.6) (54.1) (72.4) (77.3) (74.3)
4.97 3.78 3.90 3.81 4.15
(100.9) (69.8) (70.8) (73.6) (68.5)
4.55 3.92 3.92 4.05
(102.6) (53.9) (72.4) (77.3) (74.6)
5.08 4.00 4.30
(100.7) (67.4) (76.6) (80.0) (67.7)
4.53 3.90 3.90 4.01
(103.0) (53.9) (72.0) (77.5) (74.8)
5.35 4.30
(68.5)
4 GalNAc
5 GalNAc
Fuc
4.19; 4.31
(69.3)
4.70
3.82; 3.88
(69.0)
1.21
(16.9)
6 GalNAc
Fuc
3.84; 3.94
(69.2)
4.74
1.21
(16.6)
7 GalNAc
Fuc
3.90; 4.00
(69.7)
4.74
1.21
4.62
4.90
(16.9)
8 GalNAc
Fuc
3.88; 4.00
(69.0)
4.78
1.21
4.54
4.72
4.94
(98.0) (73.8) (73.8) (80.3) (67.5)
(16.9)
13
Table 3. 1H and C NMR spectral effects of sulfation and glycosylation (ꢀδ, ppm; bold
numerals indicate positions of substituents).
Compound
O-Sulfation effects
Glycosylation effects
and Residue
ꢀδH-1
ꢀδH-2
ꢀδH-3
(ꢀδC-3)
0.16
ꢀδH-4
(ꢀδC-4)
0.75
ꢀδH-5
(ꢀδC-5)
0.12
ꢀδH-6
(ꢀδC-6)
0.02
ꢀδ H-5
(ꢀδC-5)
n/a*
ꢀδ H-6
(ꢀδC-6)
n/a
(ꢀδC-1) (ꢀδC-2)
2
GalNAc
0.05
0
(0.4)
(0.6)
(-0.5)
(8.2)
(-0.3)
0.1
n/a
n/a

ACCEPTED MANUSCRI0P.2T3
3 GalNAc
4 GalNAc
6 GalNAc
Fuc
0
0
0.01
(-0.2)
0.15
(-1.1)
0.18
(0)
0.04
(-0.3)
0.74
(7.7)
0.78
(7.8)
0
n/a
n/a
n/a
n/a
0.41
(6.1)
0.38; 0.50
(6.9)
0.02; 0.06
(0.2)
0
(0)
(0)
(-2.5)
0.35
(-2.5)
0.12
(-0.7)
0.05
(-0.3)
0.17
(-0.4)
0.2
0.03
(-0.3)
0.07
(-0.4)
0
-0.01
(0.2)
0
n/a
n/a
n/a
n/a
0.18
(-1.9)
n/a
0.01; 0.11
(6.7)
n/a
(0.2)
0.02
(0.05)
0.02
(0)
0.04
(0.2)
0.2
(0.2)
0.06
(0.4)
0.11
(0)
(0.4)
0.78
(7.8)
1.09
(6.8)
0.82
(8.0)
1.13
(7.1)
0.04
(0.3)
(0.3)
0.08; 0.12
(0.7)
0
n/a
n/a
7 GalNAc
Fuc
0.23
(-1.6)
n/a
0.09; 0.19
(7.2)
n/a
(0)
0.2
0.76
(5.6)
0.18
(-0.4)
0.86
(2.8)
0.1
(-1.9)
0
(-0.8)
0.13
(-0.2)
0.2
(0)
n/a
n/a
8 GalNAc
Fuc
0.04
(0)
0.02; 0.06
(0)
0.19
(-1.4)
n/a
0.07; 0.19
(6.5)
n/a
(0)
0.38
(-2.7)
0.27
(-2.7)
0
0.74
(4.5)
0.54
(6.4)
(-0.8)
0
(0)
n/a
n/a
Fuc**
0
n/a
n/a
(-2.8)
(-0.2)
(0)
n/a
n/a
*n/a – not applicable.
** ꢀδHi = δHi (8) – δHi (7) and ꢀδCi = δCi (8) – δCi (7)
3. Conclusions
The synthesis of the selectively sulfated and non-sulfated derivatives of propyl 2-
acetamido-2-deoxy-D-galactopyranoside and 6- -α-L-fucosyl-2-acetamido-2-deoxy-D-
galactopyranoside was performed. Monosaccharides were prepared from per-O-
acetylated N-phthalimido-D-glucosamine with inversion of C-4 configuration. For the
synthesis of disaccharides , two types of glycosyl donors were studied in the
O
1-4
5-8
glycosylation reaction. Application of 2-O-benzyl-3,4-di-O-chloracetyl-L-fucopyranosyl
trichloracetimidate (16) in the presence of TMSOTf gave a mixture of α- and β-isomeric
disaccharides in a ratio of 1:1. To increase α-stereoselectivity of the reaction respective
fucosyl bromide 17 bearing equal blocking groups was applied under promotion with

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Bu4NBr. It can be supposed that the switching the mechanism of the reaction from SN1
towards SN2 led to exclusive formation of required α-isomeric glycosylation product.
Developed approach was shown to be effective and could be applied for the synthesis of
more complicated oligosaccharides related to FCS.
1
13
All the synthesized mono- and disaccharides were characterized by H and C
NMR spectroscopy to determine corresponding sulfation and glycosylation effects in their
spectra. Further conformational analysis of these oligosaccharides using nOe experiments
and theoretical modeling (quantum mechanics and molecular dynamics [20,21]) are in
progress to explain above reported NMR characteristics and to assess 3D shape FCS
chains.
4. Experimental
4.1. General procedures
Dimethylformamide (DMF, ≥99.5%) and Py (≥99.5%) were purchased from Sigma-
Aldrich. Dichloromethane (CH2Cl2) was successively distilled from diethanolamine, P2O5
and CaH2 under argon. Analytical thin-layer chromatography (TLC) was performed on
Silica Gel 60 F254 aluminium sheets (Merck), and visualization was accomplished using
UV light or by charring at ~150
chromatography was performed on Silica Gel 60, 40-63 µm (Merck). Gel
chromatography was performed on the Sephadex G-15 column (2 60 cm) by elution with
water at a flow rate of 1 mL/min and TSK HW-40S by elution with 0.1N AcOH(aq.) at a
°C with 10% (v/v) H3PO4 in ethanol. Liquid column
×
1
13
flow rate of 0.5 mL/min. H and C NMR spectra were recorded on Bruker AV400 and
1
13
AV600 spectrometers at 303 K in CDCl3 or D2O. Signal assignment in H and C NMR
1
spectra were made using COSY, TOCSY, ROESY and H–13C HSQC techniques. High-
resolution mass spectra were acquired by electrospray ionization on a MicrOTOF II

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(Bruker Daltonics) instrument. Optical rotation values were measured using a JASCO P-
2000 polarimeter at ambient temperature in solvents specified.
4.2. Standard procedures.
A - acidic O-deacetylation. A solution of AcCl (10% v/v) in MeOH was added to a
solution of saccharide in DCM. The reaction mixture was kept overnight, then neutralized
with NEt3 and concentrated in vacuo. Purification by flash column chromatography was
applied if necessary.
B – hydrogenolysis. The catalyst 10% Pd(OH)2/C was added to a stirring solution of
saccharide in an EtOAc/MeOH (1:1) mixture, and the flask was filled with H2. The
mixture was vigorously stirred under H2 (1 atm) at rt until full consumption of the starting
material and then filtered through a celite layer. The catalyst was carefully washed with
EtOAc/MeOH, and the combined filtrates were concentrated in vacuo. Purification by
flash column chromatography was applied if necessary.
C – N,O-deacylation. Hydrazine monohydrate (0.5 mL) was added to a solution of
saccharide in EtOH (5mL). The reaction mixture was kept at 90 °C for 3 h and then
concentrated in vacuo. The residue was purified by gel chromatography on the TSK HW-
40S gel with 0.1N AcOH(aq.) elution followed by lyophilization to give free amine.
D - N-acetylation. Ac2O and NaHCO3 were added to a stirring solution of free amine in
MeOH/H2O (1:1). The reaction mixture was stirred at rt overnight and then concentrated
in vacuo. The residue was purified by gel chromatography on the Sephadex G-15 gel with
water elution followed by lyophilization to give N-acetylated amine.
E – O-sulfation: Py·SO3 complex (5 eq. for each OH group) was added to a solution of
saccharide in DMF. The reaction mixture was kept at rt until the reaction was completed
(TLC) and then quenched with aqueous NaHCO3 and evaporated twice with water. The
residue was dissolved in a minimal amount of water, and then MeOH was added to

ACCEPTED MANUSCRIPT
precipitate inorganic salts. The solids were filtered off and washed with MeOH, and the
filtrate was concentrated in vacuo. Purification by flash column chromatography was
applied if necessary.
4.3. Synthesis of monosaccharide derivatives 1-4.
4.3.1. Allyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (10)
Allyl alcohol (1.5 mL, 21 mmol) was added to a stirring solution of tetraacetate 9 (5 g, 10.5
mmol) in DCM (30 mL). The mixture was cooled to 0 °C, and BF3·Et2O was added dropwise.
The reaction mixture was stirred at rt overnight, then neutralized with NEt3 (5 mL, 35 mmol),
diluted with EtOAc (250 mL), and washed with H2O (500 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was dissolved in DCM (30 mL) and treated
according to general procedure A with a 10% solution of AcCl in MeOH (10 mL).Then CSA
(300 mg, 10 mg/mL) and benzaldehyde dimethyl acetal (2.4 mL, 16 mmol) were added to the
stirring solution of the crude product in DMF (30 mL). The reaction mixture was stirred at rt
until the starting material converted to the product (1 h, TLC control, Tol–EtOAc 2:1, Rf=0.5),
then diluted with EtOAc (250 mL), and washed with H2O (500 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, Tol–EtOAc, 30:1→10:1) to give 3.15 g of the intermediate. The
latter was dissolved in DCM (30 mL), and then Py (0.7 mL, 8.7 mmol) and acetyl chloride (0.55
ml, 7.8 mmol) were added. The reaction mixture was kept at rt until full consumption of the
starting material (15 min, TLC control) and then diluted with EtOAc (250 mL) and washed with
H2O (500 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, Tol–EtOAc, 50:1→40:1) to give
monosaccharide 10 (3.24 g, 64% overall), [α]D –0.2° (c 1, EtOAc). 1H NMR (600 MHz, CDCl3):
δ 7.87 (br s, 2H, Phth), 7.74 (m, 2H, Phth), 7.46 (m, 2H, Ph), 7.36 (m, 3H, Ph), 5.90 (dd, 1H, J2-
3=10.3 Hz, J3-4=9.0 Hz, H-3), 5.75–5.62 (m, 1H, All), 5.55 (s, 1H, PhCH), 5.48 (d, 1H, J1-2=8.4

ACCEPTED MANUSCRIPT
Hz, H-1), 5.15 (dd, 1H, J=17.2 Hz, J=1.6 Hz, All), 5.06 (dd, 1H, J=10.5 Hz, J=1.3 Hz, All), 4.42
(dd, 1H, J=10.5 Hz, J5-6=4.8 Hz, H-6), 4.33 (dd, 1H, J1-2=8.4 Hz, J2-3=10.3 Hz, H-2), 4.29 (ddt,
1H, J=12.9 Hz, J=5.1 Hz, J=1.5 Hz, All), 4.05 (ddt, 1H, J=13.1 Hz, J=6.2 Hz, J=1.3 Hz, All),
3.89–3.84 (m, 1H, H-6’), 3.79 (t, 1H, J3-4=J4-5=9.2 Hz, H-4), 3.74 (td, 1H, J4-5=9.6 Hz, J5-6=4.7
Hz, H-5), 1.90 (s, 3H, Ac). 13C NMR (150 MHz, CDCl3): δ 170.2 (Ac), 134.3 (Phth), 133.2
(All), 129.2 (Ph), 128.2 (Ph), 126.2 (Ph), 123.6 (Phth), 117.9 (All), 101.7 (PhCH), 97.8 (C-1),
79.3 (C-4), 70.4 (All), 69.7 (C-3), 68.7 (C-6), 66.1 (C-5), 55.3 (C-2), 20.6 (Ac). HRMS (ESI):
Calcd m/z for [M+K]+ C26H25NO8 518.1212, found 518.1202.
4.3.2. Allyl 3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (11)
Borane trimethylamine complex (1 g, 13.3 mmol) was added to a stirring solution of
monosaccharide 10 (1.6 g, 3.33 mmol) in THF (15 mL). Then the mixture was cooled with an ice
bath, and anhydrous AlCl3 (2.7 g, 20 mmol) was added slowly. After its total dissolution H2O
(120 µL, 6.66 mmol) was added. The reaction mixture was stirred at rt until the full consumption
of the starting material (TLC control, 45 min). Then the mixture was diluted with EtOAc (150
mL) and washed with NaHCO3 (200 mL) and H2O (200 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography
(silica gel, Tol–EtOAc, 10:1→5:1) to give monosaccharide 11 (1.44 g, 90%), [α]D –9° (c 1,
EtOAc). 1H NMR (600 MHz, CDCl3): δ 7.84 (dd, 2H, J=5.4 Hz, J=3.1 Hz, Phth), 7.71 (dd, 2H,
J=5.5 Hz, J=3.0 Hz, Phth), 7.36 (d, 2H, J=4.4 Hz, Ph), 7.30–7.21 (m, 3H, Ph), 5.77–5.67 (m, 1H,
All), 5.65 (dd, 1H, J2-3=10.8 Hz, J3-4=8.8 Hz, H-3), 5.40 (d, 1H, J1-2=8.4 Hz, H-1), 5.15–5.10 (m,
1H, All), 5.05–5.00 (m, 1H, All), 4.65 (d, 1H, J=12.0 Hz, PhCHH’), 4.61 (d, 1H, J=12.0 Hz,
PhCHH’), 4.29–4.24 (m, 1H, H-2), 4.07–4.02 (m, 1H, All), 3.86 – 3.78 (m, 3H, H-6, H-4), 3.73
(dt, 1H, J4-5=9.4 Hz, J5-6=4.6 Hz, H-5), 3.05 (br s, 4-OH), 1.92 (s, 3H, Ac). 13C NMR (150 MHz,
CDCl3): δ 171.2 (Ac), 134.2 (Phth), 133.5 (All), 128.5 (Bn), 127.9 (Bn), 127.7 (Bn), 123.5
(Phth), 117.5 (All), 97.2 (C-1), 74.2 (C-5), 73.8 (PhCH2), 73.7 (C-3), 71.5 (C-4), 70.2 (C-6), 70.0

ACCEPTED MANUSCRIPT
(All), 54.6 (C-2), 20.7 (Ac). HRMS (ESI): Calcd m/z for [M+Na]+ C26H27NO8 504.1629, found
504.1625.
4.3.3. Allyl 4-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (12)
Py (1 mL, 12 mmol) was added to a stirring solution of 11 (1.44 g, 3.0 mmol) in DCM (15 mL)
under argon protection. Then the mixture was cooled to -30 °C, and Tf2O (1 mL, 6 mmol) was
added dropwise under argon protection. The reaction mixture was stirred while allowing to be
warmed from -30 °C to 10 °C over 1 h. TLC control showed full consumption of the starting
material. Then H2O (4 mL) was added, and the reaction mixture was stirred at 50 °C for 2 days.
Then the mixture was diluted with EtOAc (150 mL) and washed with H2O (300 mL). The
organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, Tol–EtOAc, 8:1→4:1) to give monosaccharide 12
1
(1.35 g, 94%), [α]D –25° (c 1, EtOAc). H NMR (400 MHz, CDCl3): δ 7.82 (dd, 2H, J=5.4 Hz,
J=3.1 Hz, Phth), 7.70 (dd, 2H, J=5.5 Hz, J=3.0 Hz, Phth), 7.40–7.22 (m, 5H, Ph), 5.78–5.63 (m,
1H, All), 5.44 (d, 1H, J3-4=3.4 Hz, H-4), 5.26 (d, 1H, J1-2=8.5 Hz, H-1), 5.12 (dd, 1H, J=17.2 Hz,
J=1.6 Hz, All), 5.04 (dd, 1H, J=10.4 Hz, J=1.4 Hz, All), 4.65 (dd, 1H, J2-3=11.1 Hz, J3-4=3.5 Hz,
H-3), 4.59 (d, 1H, J=11.9 Hz, PhCHH’), 4.49 (d, 1H, J=11.9 Hz, PhCHH’), 4.40 (dd, 1H, J2-
3=11.1 Hz, J1-2=8.5 Hz, H-2), 4.29 (ddt, 1H, J=13.0 Hz, J=5.1 Hz, J=1.5 Hz, All), 4.04 (ddt, 1H,
J=13.0 Hz, J=6.3 Hz, J=1.3 Hz, All), 3.96 (t, 1H, J5-6=6.6 Hz, H-5), 3.66–3.57 (m, 2H, H-6), 2.13
13
(s, 3H, Ac). C NMR (101 MHz, CDCl3): δ 171.6 (Ac), 134.1 (Phth), 133.5 (All), 128.5 (Bn),
127.8 (2xBn), 123.5 (Phth), 117.6 (All), 97.6 (C-1), 73.6 (PhCH2), 72.5 (C-5), 70.3 (C-4), 70.0
(All), 68.2 (C-6), 67.8 (C-3), 54.5 (C-2), 20.9 (Ac). HRMS (ESI): Calcd m/z for [M+Na]+
C26H27NO8 504.1629, found 504.1627.
4.3.4.
Allyl
3-O-benzoyl-4-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-β-D-
galactopyranoside (13)

ACCEPTED MANUSCRIPT
Py (0.25 mL, 3.0 mmol) and benzoyl chloride (0.26 ml, 2.2 mmol) were added to a solution of
12 (700 mg, 1.45 mmol) in DCM (8 mL). The reaction mixture was kept at rt until full
consumption of the starting material (6 h, TLC control), diluted with EtOAc (100 mL), and
washed with H2O (200 mL). The organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was purified by flash column chromatography (silica gel, Tol–EtOAc,
1
50:1→30:1) to give monosaccharide 13 (780 mg, 91%), [α]D 34° (c 1, EtOAc). H NMR (400
MHz, CDCl3): δ 7.84–7.76 (m, 4H, Phth, Bz), 7.71–7.62 (m, 2H, Phth), 7.52–7.43 (m, 1H, Bz),
7.39–7.23 (m, 7H, Bz, Bn), 5.96 (dd, 1H, J2-3=11.4 Hz, J3-4=3.4 Hz, H-3), 5.73 (d, 1H, J3-4=4.4
Hz, H-4), 5.79–5.67 (m, 1H, All), 5.48 (d, 1H, J1-2=8.5 Hz, H-1), 5.15 (dd, 1H, J=17.2 Hz, J=1.6
Hz, All), 5.06 (dd, 1H, J=10.4 Hz, J=1.4 Hz, All), 4.76 (dd, 1H, J2-3=11.4 Hz, J1-2=8.5 Hz, H-2),
4.60 (d, 1H, J=12.0 Hz, PhCHH’), 4.48 (d, 1H, J=12.0 Hz, PhCHH’), 4.33 (ddt, 1H, J=12.9 Hz,
J=5.1 Hz, J=1.5 Hz, All), 4.16–4.06 (m, 2H, H-5, All), 3.69–3.57 (m, 2H, H-6), 2.07 (s, 3H, Ac).
13C NMR (101 MHz, CDCl3): δ 134.2 (Phth), 133.3 (All), 133.2 (Bz), 129.6 (Bz), 128.4 (Bz,
Bn), 127.9 (Bn), 127.8 (Bn), 123.6 (Phth), 117.9 (All), 97.5 (C-1), 73.5 (PhCH2), 72.4 (C-5),
70.2 (All), 69.1 (C-3), 67.8 (C-6), 67.4 (C-4), 51.7 (C-2), 20.6 (Ac). HRMS (ESI): Calcd m/z for
[M+NH4]+ C33H31NO9 603.2337, found 603.2338.
4.3.5. Propyl 3-O-benzoyl-4-O-acetyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (14)
Hydrogenolysis of 13 (560 mg, 0.95 mmol) in EtOAc (4 mL) and MeOH (4 mL) with 10%
Pd(OH)2/C (250 mg) was performed as described in the general procedure B for 1 h. Purification
by flash column chromatography (silica gel, Tol–EtOAc, 8:1→4:1) gave product 14 (420 mg,
89%), [α]D 47° (c 1, EtOAc). 1H NMR (600 MHz, CDCl3): δ 7.88–7.77 (m, 4H, Phth, Bz), 7.72–
7.65 (m, 2H, Phth), 7.53–7.46 (m, 1H, Bz), 7.37–7.31 (m, 2H, Bz), 5.99 (dd, 1H, J2-3=11.4 Hz,
J3-4=3.5 Hz, H-3), 5.65 (d, 1H, J3-4=3.5 Hz, H-4), 5.47 (d, 1H, J1-2=8.5 Hz, H-1), 4.78 (dd, 1H, J2-
3=11.4, J3-4=8.5 Hz, H-2), 4.03 (dd, 1H, J5-6=7.1 Hz, J5-6’=6.6 Hz, H-5), 3.89–3.78 (m, 2H, Pr, H-
6), 3.64 (dd, J6-6’=11.8 Hz, J5-6’=6.6 Hz, H-6’), 3.45 (dt, 1H, J=9.7 Hz, J=6.9 Hz, Pr), 2.18 (s, 3H,

ACCEPTED MANUSCRI1P3T
Ac), 1.55–1.42 (m, 2H, Pr), 0.69 (t, 3H, J=7.4 Hz, Pr). C NMR (150 MHz, CDCl3): δ 171.1
(Bz), 165.1 (Ac), 134.2 (Phth), 133.4 (Bz), 129.6 (Bz), 128.5 (Bz), 123.4 (Phth), 98.6 (C-1), 73.7
(C-5, 71.8 (Pr), 69.0 (C-3), 67.8 (C-4), 60.8 (C-6), 51.9 (C-2), 22.6 (Pr), 20.7 (Ac), 10.1 (Pr).
HRMS (ESI): Calcd m/z for [M+Na]+ C26H27NO9 520.1578, found 520.1575.
4.3.6. Propyl 3-O-benzoyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (15)
Monosaccharide 14 (350 mg, 0.70 mmol) was dissolved in DCM (4 mL) and treated according to
the general procedure A with a 10% solution of AcCl in MeOH (2 mL). Purification by flash
column chromatography (silica gel, Tol–EtOAc, 4:1→2:1) gave product 15 (305 mg, 96%), [α]D
54° (c 1, EtOAc). 1H NMR (400 MHz, CDCl3): δ 7.91 (d, 2H, J=8.3 Hz, Bz), 7.80–7.75 (m, 2H,
Phth), 7.66 (dd, 2H, J=5.6 Hz, J=2.9 Hz, Phth), 7.52–7.43 (m, 1H, Bz), 7.34 (t, 2H, J=7.8 Hz,
Bz), 5.82 (dd, 1H, J2-3=11.4 Hz, J3-4=3.2 Hz, H-3), 5.41 (d, 1H, J1-2=8.5 Hz, H-1), 4.84 (dd, 1H,
J2-3=11.4 Hz, J1-2=8.5 Hz, H-2), 4.45 (d, 1H, J3-4=3.2 Hz, H-4), 4.08–3.95 (m, 2H, H-6), 3.90–
3.76 (m, 2H, H-5, Pr), 3.43 (dt, 1H, J=9.7 Hz, J=6.8 Hz, Pr), 1.53–1.38 (m, 2H, Pr), 0.68 (t, 3H,
J=7.4 Hz, Pr). 13C NMR (101 MHz, CDCl3): δ 165.5 (Bz), 134.2 (Phth), 133.4 (Bz), 129.8 (Bz),
128.5 (Bz), 123.5 (Phth), 98.6 (C-1), 74.1 (C-5), 71.6 (C-3, Pr), 67.7 (C-4), 62.6 (C-6), 51.3 (C-
2), 22.6 (Pr), 10.1 (Pr). HRMS (ESI): Calcd m/z for [M+Na]+ C24H25NO8 478.1472, found
478.1469.
4.3.7. Propyl 2-acetamido-2-deoxy-β-D-galactopyranoside (1)
Monosaccharide 12 (40 mg, 0.068 mmol) was successively treated according to general
procedures B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (20 mg), 1 h, flash column
chromatography (silica gel, Tol–EtOAc, 4:1→2:1)], C [N2H4·H2O (0.5 mL), EtOH (5 mL)], and
D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL), MeOH (2 mL)] to give 1 (10 mg, 56%), [α]D
5° (c 1, H2O). 1H NMR (400 MHz, D2O): δ 4.44 (d, 1H, J1-2=8.5 Hz, H-1), 3.92 (d, 1H, J3-4=3.2
Hz, H-4), 3.90–3.63 (m, 6H, H-2, Pr, H-6, H-3, H-5), 3.54 (dt, 1H, J=10.1 Hz, J=6.5 Hz, Pr),

ACCEPTED MANUSCRIPT
2.02 (s, 3H, Ac), 1.60–1.48 (m, 2H, Pr), 0.86 (t, 3H, J=7.4 Hz, Pr). 13C NMR (101 MHz, D2O): δ
176.2 (Ac), 103.0 (C-1), 76.5 (C-5), 73.6 (Pr), 72.5 (C-3), 69.3 (C-4), 62.4 (C-6), 53.9 (C-2),
23.6 (Pr) 23.5 (Ac), 11.0 (Pr). HRMS (ESI): Calcd m/z for [M+Na]+ C11H21NO6 286.1261, found
286.1254.
4.3.8. Propyl 4-O-sulfo-2-deoxy-2-phthalimido-β-D-galactopyranoside sodium salt (2)
Monosaccharide 13 (56 mg, 0.095 mmol) was successively treated according to general
procedures A [DCM (2 mL, 10% solution of AcCl in MeOH (2 mL), flash column
chromatography (silica gel, Tol–EtOAc, 10:1→4:1)], E [Py·SO3 (76 mg, 0.48 mmol), DMF (2
mL), 1 h], B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (30 mg), overnight], C [N2H4·H2O
(0.5 mL), EtOH (5 mL)], and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL), MeOH (2
mL)] to give product 2 (17 mg, 48%), [α]D –1.5° (c 1, H2O). 1H NMR (600 MHz, D2O): δ 4.68
(d, 1H, J3-4=2.0 Hz, H-4), 4.54–4.47 (m, 1H, H-1), 3.89–3.77 (m, 6H, H-2, H-3, H-5, H-6, Pr),
3.56 (dt, 1H, J=10.2 Hz, J=6.6 Hz, Pr), 2.03 (s, 3H, Ac), 1.59–1.49 (m, 2H, Pr), 0.86 (t, 3H,
13
J=7.4 Hz, Pr). C NMR(150 MHz, D2O): 176.8 (Ac), 103.4 (C-1), 77.8 (C-4).76.3 (C-5), 74.3
(Pr), 72.1 (C-3 ), 63.0 (C-6), 55.0 (C-2), 24.3 (Ac), 24.1 (Pr), 11.6 (Pr). HRMS (ESI): Calcd m/z
for [M-Na]- C11H20NO9S 342.0864, found 342.0852.
4.3.9. Propyl 6-O-sulfo-2-deoxy-2-phthalimido-β-D-galactopyranoside sodium salt (3)
Monosaccharide 14 (40 mg, 0.08 mmol) was successively treated according to general
procedures E [Py·SO3 (65 mg, 0.4 mmol), DMF (2 mL), 1 h], C [N2H4·H2O (0.5 mL), EtOH (5
mL)], and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL), MeOH (2 mL)] to give product 3
(15 mg, 52%), [α]D 2.4° (c 1, H2O). 1H NMR (400 MHz, D2O): δ 4.46 (d, 1H, J1-2=8.5 Hz, H-1),
4.25–4.14 (m, 2H, H-6), 3.98 (d, 1H, J3-4=3.3 Hz, H-4), 3.93–3.87 (m, 2H, H-2, H-5), 3.87–3.79
(m, 1H, Pr), 3.73 (dd, 1H, J2-3=10.8 Hz, J3-4=3.3 Hz, H-3), 3.56 (dt, 1H, J=10.2 Hz, J=6.5 Hz,
13
Pr), 2.02 (s, 3H, Ac), 1.61–1.48 (m, 2H, Pr), 0.86 (t, 3H, J=7.4 Hz, Pr). C NMR (101 MHz,

ACCEPTED MANUSCRIPT
D2O): δ 176.2 (Ac), 103.0 (C-1), 74.0 (C-5), 73.8 (Pr), 72.3 (C-3), 69.0 (C-4), 68.5 (C-4), 53.8
(C-2), 23.6 (Pr), 23.5 (Ac), 11.0 (Pr). HRMS (ESI): Calcd m/z for [M-Na]- C11H20NO9S
342.0864, found 342.0864.
4.3.10. Propyl 4,6-di-O-sulfo-2-deoxy-2-phthalimido-β-D-galactopyranoside sodium salt (4)
Monosaccharide 15 (77 mg, 0.169 mmol) was successively treated according to general
procedures E [Py·SO3 (270 mg, 1.7 mmol), DMF (2 mL), 2 h], C [N2H4·H2O (0.5 mL), EtOH (5
mL)], and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL), MeOH (2 mL)] to give 4 (39 mg,
48%). [α]D 6° (c 1, H2O). 1H NMR (600 MHz, D2O): δ 4.67 (d, 1H, J3-4=1.4 Hz, H-4), 4.50–4.46
(m, 1H, H-1), 4.27 (dd, 1H, J6-6’=11.5 Hz, J5-6=3.5 Hz, H-6), 4.16 (dd, 1H, J6-6’=11.5 Hz, J5-
6’=8.4 Hz, H-6’), 4.03–3.99 (m, 1H, H-5), 3.85–3.82 (m, 2H, H-2, H-3), 3.81 (dt, 1H, J=10.2 Hz,
J=6.3 Hz, Pr), 3.54 (dt, 1H, J=10.2 Hz, J=6.6 Hz, Pr), 1.98 (s, 3H, Ac), 1.51 (dd, 2H, J=13.9 Hz,
J=6.6 Hz, Pr), 0.82 (t, 3H, J=7.4 Hz, Pr). 13C NMR (151 MHz, D2O): δ 176.2 (Ac), 102.7 (C-1),
77.0 (C-4), 73.8 (Pr), 73.5 (C-5), 71.4 (C-3), 69.2 (C-6), 54.1 (C-2), 23.6 (Ac), 23.5 (Pr), 11.0
(Pr). HRMS (ESI): Calcd m/z for [M-Na]- C11H19NO12S2Na 444.0252, found 444.0252.
4.4. Synthesis of disaccharide derivatives 5-8.
4.4.1. 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranose bromide (17).
0.1 M TMSOTf in DCM (250 ꢁL) was added to a stirred solution of donor 16 (565 mg, 1.02
mmol) in wet DCM (7 mL), and the mixture was stirred for 30 min. Then the mixture was
neutralized with Et3N and evaporated in vacuo. The residue was purified by flash column
chromatography (silica gel, eluent: toluene/EtOAc = 10:1→2:1). The resulting hemiacetal was
dissolved in DCM (5 mL), tetrabromomethane (815 mg, 2.46 mmol) and triphenylphosphine
(645 mg, 2.46 mmol) were added, and the mixture was stirred at rt for 30 min and then purified
by column chromatography (eluent: toluene/EtOAc = 50:1→30:1) to give bromide 17 (410 mg,
1
85%), [α]D –70° (c 1, EtOAc). H NMR (400 MHz, CDCl3): δ 7.39–7.29 (m, 5H, Bn), 6.10 (t,

ACCEPTED MANUSCRIPT
1H, J1-2=4.5 Hz, H-1), 5.46–5.33 (m, 2H, H-3, H-4), 4.72–4.66 (m, 2H, PhCHH’), 4.51–4.40 (m,
1H, H-5), 4.11 (d, 1H, J=5.8 Hz, CHH’Cl), 4.06 (dd, 1H, J2-3=10.2 Hz, J1-2=3.9 Hz, H-2), 3.97
(d, 1H, J = 1.3 Hz, CHH’Cl), 3.92–3.80 (m, 1H, CHH’Cl), 3.79–3.75 (m, 1H, CHH’Cl), 1.20 (d,
3H, J5-6=6.6 Hz, H-6). 13C NMR (101 MHz, CDCl3): δ 167.1 (CA), 128.6 (Bn), 128.3 (Bn),
128.0 (Bn), 93.3 (C-1), 73.0 (Bn), 72.5 (C-2), 72.1 (C-4), 71.50 (C-3), 67.5 (C-5), 40.4 (CA),
24.9 (CA), 15.60 (C-6). HRMS (ESI): Calcd m/z for [M+NH4]+ C17H19BrCl2O6 486.0080, found
486.0082.
4.4.2.
Propyl
6-O-(2-O-benzyl-3,4-di-O-chloroacetyl-L-fucopyranosyl)-3-O-benzoyl-2-
deoxy-2-phthalimido-β-D-galactopyranoside (18αβ)
0.1 M TMSOTf in DCM (30 ꢁL) was added to a mixture of glycosyl donor 16 (61 mg, 0.11
mmol), glycosyl acceptor 15 (42 mg, 0.09 mmol), and molecular sieves 4 Å (100 mg) in DCM (1
mL) at -50 °C under argon protection. The mixture was stirred for 30 min, then neutralized with
Et3N, and subjected to column chromatography (eluent: toluene/EtOAc = 20:1→5:1) to give
1
glycosylation product 18αβ (39 mg, 0.046 mmol, 51%) as a mixture of isomers (α:β=1:1). H
NMR (600 MHz, CDCl3): δ 7.94–7.89 (m, 4H, Bzα, Bzβ), 7.81–7.79 (m, 4H, Phthα, Phthβ), 7.69–
7.65 (m, 4H, Phthα, Phthβ), 7.53–7.47 (m, 2H, Bzα, Bzβ), 7.39–7.15 (m, 14H, Bzα, Bzβ, Bnα, Bnβ),
5.87 (dd, 1H, J2-3=11.3 Hz, J3-4=3.2 Hz, H-3 (GalNα)), 5.83 (dd, 1H, J2-3=11.4 Hz, J3-4=3.2 Hz,
H-3 (GalNβ)), 5.41–5.33 (m, 4H, H-1 (GalNα), H-1 (GalNβ), H-3 (Fucα), H-4 (Fucα)), 5.26 (d,
1H, J3-4=3.4 Hz, H-4 (Fucβ)), 5.07 (dd, 1H, J2-3=10.1 Hz, J3-4=3.4 Hz, H-3 (Fucβ)), 4.88 (d, 1H,
J1-2=3.7 Hz, H-1 (Fucα)), 4.85–4.76 (m, 3H, H-2 (GalNα), H-2 (GalNβ), PhCHH’β), 4.72–4.60
α
(m, 3H, PhCHH’β, PhCH2 ), 4.55 (d, 1H, J1-2=7.7 Hz, H-1 (GalNβ)), 4.40 (d, 2H, J3-4=2.9 Hz, H-
4 (GalNα), H-4 (GalNβ)), 4.24 (q, 1H, J5-6=6.5 Hz, H-5 (Fucα)), 4.18–4.09 (m, 5H, H-6 (GalNβ),
CAα, CAβ), 4.06–3.97 (m, 4H, H-5 (GalNα), H-5 (GalNβ), H-6’ (GalNβ), H-6 (GalNα)), 3.96–3.90
(m, 4H, CAα, CAβ), 3.89–3.83 (2H, m, H-6’ (GalNα), H-2 (Fucα)), 3.81–3.76 (m, 3H, H-5 (Fucβ),
Prα, Prβ), 3.73 (dd, 1H, J6-6’=10.1 Hz, J5-6=5.2 Hz, H-6’(GalNα)), 3.64 (dd, 1H, J2-3=10.1 Hz, J1-

ACCEPTED MANUSCRIPT
2=7.7 Hz, H-2 (Fucβ)), 3.42–3.37 (m, 2H, Prα, Prβ), 1.50 – 1.39 (m, 4H, Prα, Prβ), 1.26 (d, 3H, J5-
6=6.4 Hz, H-6 (Fucβ)), 1.15 (d, 3H, J5-6=6.6 Hz, H-6 (Fucα)), 0.68 (t, 6H, J=7.4 Hz, Prα, Prβ). 13C
NMR (151 MHz, CDCl3): δ 134.1 (Phthα, Phthβ ), 133.4 (Bzα, Bzβ), 129.9 (Bzα), 129.8 (Bzβ),
128.5 (2xBnα, 2xBnβ), 128.3 (Bzα, Bzβ), 127.9 (Bnβ), 127.8 (Bnα), 123.4 (Phthα, Phthβ), 103.6 (C-
1 (Fucβ)), 98.6 (C-1 (GalNβ)), 98.5 (C-1 (GalNα)), 97.9 (C-1 (Fucα)), 75.9 (C-2 (Fucβ)), 74.9
(Bnβ)), 74.2 (C-3 (Fucβ)), 73.2 (C-4 (Fucα), Bnα), 72.9 (C-2 (Fucα)), 72.6 (C-5 (GalNβ)), 72.5 (C-
5 (GalNα)), 72.4 (C-4 (Fucβ)), 71.8 (C-3 (Fucα)), 71.5 (C-3 (GalNβ)), 71.4 (Prα, Prβ),71.3 (C-3
(GalNα)), 68.7 (C-5 (Fucβ)), 67.9 (C-6 (GalNβ)), 67.2 (C-4 (GalNβ)), 67.0 (C-6 (GalNα)), 66.6
(C-4 (GalNα)), 64.3 (C-5 (Fucα)), 51.4 (C-2 (GalNα), C-2 (GalNβ)), 40.5 (CAα, CAβ), 40.4 (CAα,
CAβ), 22.6 (Prα, Prβ), 16.1 (C-6 (Fucβ)), 15.8 (C-6 (Fucα)), 10.1 (Prα, Prβ). HRMS (ESI): Calcd
m/z for [M+Na]+ C41H43Cl2NO14 866.1953, found 866.1957.
4.4.3. Propyl 6-O-(2-O-benzyl-3,4-di-O-chloroacetyl-L-fucopyranosyl)-3-O-benzoyl-4-O-
acetyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (19αβ).
0.1 M TMSOTf in DCM (100 ꢁL) was added to a mixture of glycosyl donor 16 (240 mg, 0.43
mmol), glycosyl acceptor 14 (180 mg, 0.36 mmol), and molecular sieves 4 Å (300 mg) in DCM
(3 mL) at -50 °C under argon protection. The mixture was stirred for 30 min, then neutralized
with Et3N, and purified by column chromatography (eluent: toluene/EtOAc = 20:1→10:1) to
1
give glycosylation product 19αβ (250 mg, 78%) as a mixture of isomers (α:β=2:1). H NMR
(600 MHz, CDCl3): δ 7.92 (d, 2H, J=8.3 Hz, Bzβ), 7.86–7.76 (m, 6H, Bzα, Phthα, Phthβ), 7.70–
7.63 (m, 4H, Phthα, Phthβ), 7.53–7.43 (m, 2H, Bzα, Bzβ), 7.40–7.22 (m, 14H, Bzα, Bzβ, 5xBnα,
5xBnβ), 6.01–5.94 (m, 1H, H-3 (GalNα)), 5.85–5.81 (m, 1H, H-3 (GalNβ)), 5.71 (d, 1H, J3-4=3.6
Hz, H-4 (GalNα)), 5.64 (d, 1H, J3-4=3.5 Hz, H-4 (GalNβ)), 5.50–5.37 (m, 3H, H-1 (GalNα), H-1
(GalNβ), H-3 (Fucα)), 5.35–5.31 (m, 1H, H-4 (Fucα)), 5.25 (d, 1H, J3-4=3.4 Hz, H-4 (Fucβ)), 5.04
(dd, 1H, J2-3=10.1 Hz, J3-4=3.4 Hz, H-3 (Fucβ)), 4.89 (d, 1H, J1-2=3.7 Hz, H-1 (Fucα)), 4.83–4.72
α
(m, 3H, H-2 (GalNα), H-2 (GalNβ), PhCHH’β), 4.71–4.63 (m, 2H, PhCH2 ), 4.58 (d, 1H, J=11.9

ACCEPTED MANUSCRIPT
Hz, PhCHH’β), 4.49 (d, 1H, J1-2=7.7 Hz, H-1 (Fucβ)), 4.19–4.08 (m, 6H, H-5 (GalNα), H-5
(Fucα), CAα, CAβ), 4.04–4.00 (m, 2H, H-5 (GalNβ), H-6 (GalNβ)), 3.97–3.91 (m, 4H, CAα, CAβ),
3.89–3.76 (m, 6H, H-2 (Fucα), H-6’ (GalNβ), H-5 (Fucβ), H-6 (GalNα), Prα, Prβ), 3.67 (dd, 1H, J6-
6'=10.5 Hz, J5-6=6.4 Hz, H-6' (GalNα)), 3.61 (dd, 1H, J2-3=10.2 Hz, J1-2=7.6 Hz, H-2 (Fucβ)),
3.48–3.37 (m, 2H, Prα, Prβ), 2.17 (s, 3H, Acβ), 2.16 (s, 3H, Acα), 1.53–1.37 (m, 4H, Prα, Prβ),
1.26 (d, 3H, J5-6=6.7 Hz, H-6 (Fucβ)), 1.12 (d, 3H, J5-6=6.6 Hz, H-6 (Fucα)), 0.74–0.60 (m, 6H,
13
Prα, Prβ). C NMR (151 MHz, CDCl3): δ 167.2 (Bz), 165.0 (Ac), 134.2 (Phthα, Phthβ), 133.3
(Bzα, Bzβ), 129.8 (Bnβ), 129.6 (Bzα, Bzβ), 128.5 (Bnα), 128.4 (Bzα, Bzβ), 128.3 (Bnβ), 128.0 (Bnα,
Bnβ), 127.7 (Bnα), 123.4 (Phthα, Phthβ), 103.4 (C-1 (Fucβ)), 98.6 (C-1 (GalNβ)), 98.5 (C-1
(GalNα)), 98.2 (C-1 (Fucα)), 75.8 (C-2 (Fucβ)), 74.7 (Bnβ), 74.0 (C-3 (Fucβ)), 73.7 (C-5 (GalNβ)),
73.2 (C-4 (Fucα)), 73.0 (C-2 (Fucα)), 72.9 (Bnα), 72.3 (C-4 (Fucβ)), 72.2 (C-5 (GalNα)), 71.7
(Prβ), 71.6 (Prα), 71.5 (C-3 (Fucα)), 69.0 (C-3 (GalNα)), 68.6 (C-5 (Fucβ)), 67.8 (C-4 (GalNβ)),
67.6 (C-4 (GalNα)), 67.3 (C-6 (GalNα), C-3 (GalNβ)), 67.2 (C-6 (GalNβ)), 64.4 (C-5 (Fucα)), 51.9
(C-2 (GalNβ)), 51.8 (C-2 (GalNα)), 40.5 (CAα), 40.4 (CAβ), 22.6 (Prα, Prβ), 20.6 (Acα, Acβ), 16.1
(C-6 (Fucβ)), 15.9 (C-6 (Fucα)), 10.1 (Prα, Prβ). HRMS (ESI): Calcd m/z for [M+K]+
C43H45Cl2NO15 924.1798, found 924.1794.
4.4.4. Propyl 6-O-(2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl)-3-O-benzoyl-2-
deoxy-2-phthalimido-β-D-galactopyranoside (18α)
TBAB (65 mg, 0.15 mmol) was added to a mixture of glycosyl donor 16 (260 mg, 0.55 mmol)
and glycosyl acceptor 15 (200 mg, 0.44 mmol) in DCM (4 mL). The mixture was kept for 7 days
and then subjected to column chromatography (eluent: toluene/EtOAc = 20:1→2:1) to give a
mixture of 15 and 16 (230 mg) and glycosylation product 18α (104 mg, 56%), [α]D –44° (c 1,
1
EtOAc). H NMR (400 MHz, CDCl3): δ 7.93 (d, 1H, J=8.3 Hz, Bz), 7.80 (dd, 2H, J=5.2 Hz,
J=3.3 Hz, Phth), 7.67 (dd, 2H, J=5.6 Hz, J=2.9 Hz, Phth), 7.54–7.46 (m, 1H, Bz), 7.42–7.22 (m,
7H, Bz, Bn), 5.87 (dd, 1H, J2-3=11.3 Hz, J3-4=3.2 Hz, H-3 (GalN)), 5.40 (d, 1H, J1-2=8.5 Hz, H-1

ACCEPTED MANUSCRIPT
(GalN)), 5.39–5.28 (m, 2H, H-3 (Fuc), H-4 (Fuc)), 4.88 (t, 1H, J1-2=4.4 Hz, H-1 (Fuc)), 4.81 (dd,
1H, J2-3=11.3 Hz, J1-2=8.5 Hz, H-2 (GalN)), 4.76–4.65 (m, 2H, PhCH2), 4.41 (br s, 1H, H-4
(GalN)), 4.24 (q, 1H, J5-6=5.9 Hz, H-5 (Fuc)), 4.12 (d, 2H, J=5.6 Hz, CA), 4.06–3.97 (m, 2H, H-
5 (GalN), H-6 (GalN)), 3.96 – 3.70 (m, 5H, CA, H-2 (Fuc), Pr, H-6’ (GalN)), 3.40 (dt, 1H, J=9.6
Hz, J=6.8 Hz, Pr), 2.61 (br s, 1H,4-OH (GalN)), 1.50–1.38 (m, 2H, Pr), 1.15 (d, 3H, J5-6=6.5 Hz,
H-6 (Fuc)), 0.67 (t, 3H, J=7.4 Hz, Pr). 13C NMR (101 MHz, CDCl3): δ 167.3 (Bz), 165.3 (CA),
134.1 (Phth), 133.4 (Bz), 129.9 (Bz), 128.5 (2xBn), 128.5 (Bz), 128.1 (Bn), 123.5 (Phth), 98.5
(C-1 (GalN)), 97.9 (C-1 (Fuc)), 73.30 (PhCH2), 73.0 (C-2 (Fuc)), 72.7 (C-4 (Fuc)), 72.4 (C-5
(GalN)), 72.0 (C-3 (Fuc)), 71.3 (C-3 (GalN), Pr), 67.1 (C-4 (GalN)), 67.0 (C-6 (GalN)), 64.3 (C-
5 (Fuc)), 51.3 (C-2 (GalN)), 40.5 (CA), 25.1 (CA), 22.6 (Pr), 15.8 (C-6 (Fuc)), 10.2 (Pr). HRMS
(ESI): Calcd m/z for [M+Na]+ C41H43Cl2NO14 866.1953, found 866.1955.
4.4.5. Propyl 6-O-(2-O-benzyl-α-L-fucopyranosyl)-3-O-benzoyl-2-deoxy-2-phthalimido-β-
D-galactopyranoside (20).
A mixture of disaccharide 18α (35 mg, 0.041 mmol), 2,4,6-collidine (28 ꢁL, 0.207 mmol), and
thiourea (50 mg, 0.62 mmol) in MeOH (4 mL) was refluxed for 24 h at 60 °C, then cooled, and
taken to dryness. The residue was purified by flash column chromatography (silica gel, eluent:
1
toluene/EtOAc = 3:1→1:1) to give 21 (26.5 mg, 92%), [α]D –28° (c 1, EtOAc). H NMR (600
MHz, CDCl3): δ 7.92 (d, 2H, J=8.3 Hz, Bz), 7.82–7.77 (m, 2H, Phth), 7.67 (dd, 2H, J=5.5, J=3.0
Hz, Phth), 7.51–7.45 (m, 1H, Bz), 7.38–7.30 (m, 7H, Bz, Bn), 5.82 (dd, 1H, J2-3=11.3 Hz, J3-
4=3.2 Hz, H-3 (GalN)), 5.38 (d, 1H, J1-2=8.5 Hz, H-1 (GalN)), 4.85 (dd, 1H, J2-3=11.3 Hz, J1-
2=8.5 Hz, H-2 (GalN)), 4.82 (d, 1H, J1-2=3.6 Hz, H-1 (Fuc)), 4.67 (d, 1H, J=12.0 Hz, PhCHH’),
4.64 (d, 1H, J=12.0 Hz, PhCHH’), 4.43 (d, 1H, J3-4=3.2 Hz, H-4 (GalN)), 4.05–3.98 (m, 3H, H-5
(Fuc), H-3 (Fuc), H-6 (GalN)), 3.92 (t, 1H, J5-6=5.3 Hz, H-5 (GalN)), 3.81 (d, 1H, J3-4=3.4 Hz,
H-4 (Fuc)), 3.78 (dt, 1H, J=9.7 Hz, J=6.5 Hz, Pr), 3.72 (dd, 1H, J2-3=9.8 Hz, J1-2=3.6 Hz, H-2
(Fuc)), 3.64 (dd, 1H, J6-6’=10.5 Hz, J5-6=5.0 Hz, H-6’ (GalN)), 3.40 (dt, 1H, J=9.7 Hz, J=6.8 Hz,

ACCEPTED MANUSCRIPT
13
Pr), 1.50–1.38 (m, 2H, Pr), 1.27 (d, 3H, J5-6=6.6 Hz, H-6 (Fuc)), 0.68 (t, 3H, J=7.4 Hz, Pr). C
NMR (151 MHz, CDCl3): δ 165.5 (Bz), 134.1 (Phth), 133.3 (Bz), 129.8 (Bz), 128.6 (Bn), 128.4
(Bz, Bn), 128.1 (Bn), 123.4 (Phth), 98.6 (C-1 (GalN)), 97.2 (C-1 (Fuc)), 76.5 (C-2 (Fuc)), 72.9
(PhCH2) , 72.5 (C-5 (GalN)), 71.6 (C-4 (Fuc)), 71.5 (C-3 (GalN)), 71.3 (Pr), 69.4 (C-3 (Fuc)),
67.4 (C-4 (GalN)), 67.2 (C-6 (GalN)), 65.8 (C-5 (Fuc)), 51.3 (C-2 (GalN)), 22.6 (Pr), 16.1 (C-6
(Fuc)), 10.1 (Pr). HRMS (ESI): Calcd m/z for [M+Na]+ C37H41NO12 714.2521, found 714.2515.
4.4.6. Propyl 6-O-(α-L-fucopyranosyl)-2-acetamido-2-deoxy-β-D-galactopyranoside (5)
Disaccharide 18α (36 mg, 0.043 mmol) was successively treated according to general procedures
B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (30 mg), 3 h], C [N2H4·H2O (0.5 mL), EtOH
(5 mL)] and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL), MeOH (2 mL)] to give 5 (9 mg,
1
51%), [α]D –25° (c 1, H2O). H NMR (600 MHz, D2O): δ 4.96 (d, 1H, J1-2=3.9 Hz, H-1 (Fuc)),
4.48 (d, 1H, J1-2=8.5 Hz, H-1 (GalN)), 4.10 (q, 1H, J5-6=6.7 Hz, H-5 (Fuc)), 3.96 (d, 1H, J3-4=3.3
Hz, H-4 (GalN)), 3.92–3.77 (m, 8H, H-6 (GalN), H-6’ (GalN), H-2 (GalN), H-5 (GalN), H-3
(Fuc), H-4 (Fuc), Pr, H-2 (Fuc)), 3.74 (dd, 1H, J2-3=10.8 Hz, J3-4=3.3 Hz, H-3 (GalN)), 3.56 (dt,
1H, J=10.0 Hz, J=6.6 Hz, Pr), 2.05 (s, 3H, Ac), 1.60–1.52 (m, 2H, Pr), 1.24 (d, 1H, J5-6=6.6 Hz,
H-6 (Fuc)), 0.88 (t, 3H, J=7.4 Hz, Pr). 13C NMR(151 MHz, CDCl3): δ 103.0 (C-1 (GalN)), 100.7
(C-1 (Fuc)), 74.9 (C-5 (GalN)), 73.7 (Pr), 73.1 (C-4 (Fuc)), 72.4 (C-3 (GalN)), 70.9 (C-3 (Fuc)),
69.4 (C-2 (Fuc)), 69.3 (C-4 (GalN)), 69.0 (C-6 (GalN)), 68.2 (C-5 (Fuc)), 53.7 (C-2 (GalN)),
23.6 (Ac), 23.5 (Pr), 16.8 (C-6 (Fuc)), 11.0 (Pr)). HRMS (ESI): Calcd m/z for [M+Na]+
C17H31NO10 432.1840, found 432.1828.
4.4.7. Propyl 6-O-(α-L-fucopyranosyl)-4-O-sulfo-2-deoxy-2-acetyl-β-D-galactopyranoside
sodium salt (6).
Disaccharide 18α (20 mg, 0.024 mmol) was successively treated according to general procedures
E [Py·SO3 (20 mg, 0.12 mmol), DMF (2 mL), 1 h, flash column chromatography (silica gel,

ACCEPTED MANUSCRIPT
DCM–MeOH, 10:1→2:1)], B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (30 mg), 2 days],
C [N2H4·H2O (0.5 mL), EtOH (5 mL)] and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL),
MeOH (2 mL)] to give 6 (5 mg, 42%), [α]D –48° (c 1, H2O). 1H NMR (600 MHz, D2O): δ 4.95
(d, 1H, J1-2=3.9 Hz, H-1 (Fuc)), 4.71 (s, 1H, H-4 (GalN), srp), 4.56–4.50 (m, 1H, H-1 (GalN)),
4.11 (q, 1H, J5-6=6.6 Hz, H-5 (Fuc)), 3.98 (dd, 1H, J5-6’=8.3 Hz, J5-6=3.9 Hz, H-5 (GalN)), 3.91
(dd, 1H, J6-6’=11.7 Hz, J5-6=3.9 Hz, H-6 (GalN)), 3.88–3.78 (m, 6H, H-2 (GalN), H-3 (GalN), H-
3 (Fuc), Pr, H-6’ (GalN), H-4 (Fuc)), 3.75 (dd, 1H, J2-3=10.4 Hz, J1-2=3.9 Hz, H-2 (Fuc)), 3.55
(dt, 1H, J=10.0 Hz, J=6.6 Hz, Pr), 2.03 (s, 3H, Ac), 1.59–1.51 (m, 2H, Pr), 1.22 (d, 3H, J5-6=6.6
Hz, H-6 (Fuc)), 0.86 (t, 3H, J=7.4 Hz, Pr). 13C NMR (151 MHz, D2O): δ 176.0 (Ac), 102.6 (C-1
(GalN)), 100.9 (C-1 (Fuc)), 77.3 (C-4 (GalN)), 74.3 (C-5 (GalN)), 73.6 (Pr), 73.0 (C-4 (Fuc)),
71.3 (C-3 (GalN)), 70.8 (C-3 (Fuc)), 69.3 (C-2 (Fuc)), 69.2 (C-6 (GalN)), 68.0 (C-5 (Fuc)), 54.1
(C-2 (GalN)), 23.4 (Pr), 23.3 (Ac), 16.7 (C-6 (Fuc)), 10.8 (Pr). HRMS (ESI): Calcd m/z for [M-
Na]- C17H30NO13S 488.1443, found 488.1436.
4.4.8.
Propyl
6-O-(3,4-di-O-sulfo-α-L-fucopyranosyl)-4-O-sulfo-2-deoxy-2-acetyl-β-D-
galactopyranoside sodium salt (7)
Disaccharide 20 (16 mg, 0.023 mmol) was successively treated according to general procedures
E [Py·SO3 (55 mg, 0.35 mmol), DMF (2 mL), 1h, flash column chromatography (silica gel,
DCM – MeOH, 10:1→2:1)], B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (30 mg), 1 day],
C [N2H4·H2O (0.5 mL), EtOH (5 mL)] and D [Ac2O (0.4 mL, NaHCO3 (340 mg), H2O (2 mL),
MeOH (2 mL)] to give 7 (6.2 mg, 37%), [α]D –66° (c 1, H2O). 1H NMR (600 MHz, D2O): δ 4.93
(d, 1H, J1-2=3.9 Hz, H-1 (Fuc)), 4.76 (d, 1H, J3-4=3.0 Hz, H-4 (Fuc)), 4.59 (d, 1H, J3-4=2.8 Hz, H-
4 (GalN)), 4.46 (dd, 1H, J2-3=10.4 Hz, J3-4=3.0 Hz, H-3 (Fuc)), 4.41 (d, 1H, J1-2=7.8 Hz, H-1
(GalN)), 4.14 (q, 1H, J5-6=6.6 Hz, H-5 (Fuc)), 3.90 (dd, 1H, J5-6=8.6 Hz, J5-6’=3.3 Hz, H-5
(GalN)), 3.87–3.82 (m, 2H, H-2 (Fuc), H-6 (GalN)), 3.79–3.70 (m, 4H, H-2 (GalN), H-3 (GalN),
H-6’ (GalN), Pr), 3.45 (dt, 1H, J=10.1 Hz, J=6.6 Hz, Pr), 1.91 (s, 3H, Ac), 1.44 (dd, 2H, J=13.9

ACCEPTED MANUSCRIPT
13
Hz, J=6.7 Hz, Pr), 1.17 (d, 3H, J5-6=6.6 Hz, H-6 (Fuc)), 0.75 (t, 3H, J=7.4 Hz, Pr). C NMR
(151 MHz, D2O): δ 102.6 (C-1 (GalN)), 100.7 (C-1 (Fuc)), 80.2 (C-4 (Fuc)), 77.3 (C-4 (GalN)),
76.6 (C-3 (Fuc)), 74.6 (C-5 (GalN)), 73.6 (Pr), 71.3 (C-3 (GalN)), 69.6 (C-6 (GalN)), 67.7 (C-5
(Fuc)), 67.4 (C-2 (Fuc)), 54.1 (C-2 (GalN)), 23.3 (Pr, Ac), 17.3 (C-6 (Fuc)), 10.8 (Pr). HRMS
(ESI): Calcd m/z for [M-2Na]2- C17H28NO19S3Na 334.5163, found 334.5154.
4.4.9. Propyl 6-O-(2,3,4-tri-O-sulfo-α-L-fucopyranosyl)-4-O-sulfo-2-acetamido-2-deoxy-β-
D-galactopyranoside sodium salt (8)
Disaccharide 20 (26 mg, 0.037 mmol) was successively treated according to general procedures
B [EtOAc (1mL), MeOH (1 mL), 10% Pd(OH)2/C (40 mg), 2 h], E [Py·SO3 (120 mg, 0.75
mmol), DMF (2 mL), 2 h], C [N2H4·H2O (0.5 mL), EtOH (5 mL)] and D [Ac2O (0.4 mL,
NaHCO3 (340 mg), H2O (2 mL), MeOH (2 mL)] to give 8 (14.3 mg, 47%), [α]D –54° (c 1, H2O).
1H NMR (600 MHz, D2O): δ 5.43 (d, 1H, J1-2=3.7 Hz, H-1 (Fuc)), 5.03 (d, 1H, J3-4=3.1 Hz, H-4
(Fuc)), 4.86 (d, 1H, J3-4=2.2 Hz, H-4 (GalN))), 4.81 (dd, 1H, J2-3=10.6 Hz, J3-4=3.1 Hz, H-3
(Fuc)), 4.65–4.59 (m, 2H, H-2 (Fuc), H-1 (GalN)), 4.39 (q, 1H, J5-6=6.6 Hz, H-5 (Fuc)), 4.12–
4.06 (m, 2H, H-5 (GalN), H-6 (GalN)), 4.00–3.92 (m, 4H, H-2 (GalN), H-3 (GalN), H-6’
(GalN), Pr), 3.66 (dt, 1H, J=10.1 Hz, J=6.5 Hz, Pr), 2.13 (s, 3H, Ac), 1.69–1.62 (m, 2H, Pr), 1.40
13
(d, 3H, J5-6=6.6 Hz, H-6 (Fuc)), 0.97 (t, 3H, J=7.4 Hz, Pr). C NMR (151 MHz, D2O): δ 176.0
(Ac), 102.6 (C-1 (GalN)), 98.6 (C-1 (Fuc)), 80.4 (C-4 (Fuc)), 77.5 (C-4 (GalN)), 74.4 (C-5
(GalN)), 73.8 (C-3 (Fuc)), 73.6 (Pr), 73.5 (C-2 (Fuc)), 71.4 (C-3 (GalN)), 69.0 (C-6 (GalN)),
67.6 (C-5 (Fuc)), 54.1 (C-2 (GalN)), 23.5 (Pr), 23.3 (Ac), 17.2 (C-6 (Fuc), 10.9 (Pr). HRMS
(ESI): Calcd m/z for [M-Na]- C17H27NO22S4Na3 793.9606, found 793.9611.
Acknowledgements
This work was supported by the Russian Science Foundation (grant 14-13-01325).

ACCEPTED MANUSCRIPT
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