Synthesis of 3-aminopropyl glycosides of linear β-(1→3)-d-glucooligosaccharides

Article abstract of DOI:10.1016/j.carres.2015.10.012

3-Aminopropyl glycosides of a series of linear β-(1→3)-linked d-glucooligosaccharides containing from 3 to 13 monosaccharide units were efficiently prepared. The synthetic scheme featured highly regioselective glycosylation of 4,6-O-benzylidene-protected 2,3-diol glycosyl acceptors with a disaccharide thioglycoside donor bearing chloroacetyl groups at O-2′ and -3′ as a temporary protection of the diol system. Iteration of the deprotection and glycosylation steps afforded the series of the title oligoglucosides differing in length by two monosaccharide units. A novel procedure for selective removal of acetyl groups in the presence of benzoyl ones consisting in a brief treatment with a large excess of hydrazine hydrate has been proposed.

Full text of DOI:10.1016/j.carres.2015.10.012

Carbohydrate Research 419 (2016) 1–10
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of 3-aminopropyl glycosides of linear
β-(1→3)-d-glucooligosaccharides
Dmitry V. Yashunsky a, Yury E. Tsvetkov a, Alexey A. Grachev a, Alexander O. Chizhov b,
Nikolay E. Nifantiev a,*
a Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991
Moscow, Russia
b Division of Structural Studies, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russia
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 13 September 2015
Received in revised form 18 October 2015
Accepted 23 October 2015
Available online 30 October 2015
3-Aminopropyl glycosides of a series of linear β-(1→3)-linked d-glucooligosaccharides containing from
3 to 13 monosaccharide units were efficiently prepared. The synthetic scheme featured highly regioselective
glycosylation of 4,6-O-benzylidene-protected 2,3-diol glycosyl acceptors with a disaccharide thioglycoside
donor bearing chloroacetyl groups at O-2′ and -3′ as a temporary protection of the diol system. Itera-
tion of the deprotection and glycosylation steps afforded the series of the title oligoglucosides differing
in length by two monosaccharide units. A novel procedure for selective removal of acetyl groups in the
presence of benzoyl ones consisting in a brief treatment with a large excess of hydrazine hydrate has
been proposed.
Keywords:
β-(1→3)-Glucooligosaccharides
β-Glucan
© 2015 Elsevier Ltd. All rights reserved.
De-O-acetylation
1. Introduction
3-aminopropyl glycosides for further preparation of conjugated
vaccine candidates, as well as labeled oligosaccharides and other
β-(1→3)-Glucans are widespread in Nature being essential con-
stituents of the cell wall in fungi and yeasts1,2 and a major storage
polysaccharide in brown seaweeds.3 Great interest in this type of poly-
saccharides relates to their immunostimulating properties, including
antibacterial and antitumor activities.4,5 On the other hand, the pres-
ence of highly conserved β-(1→3)-glucans in different pathogenic fungal
species makes these glycopolymers a rational target for vaccine devel-
opment. Conjugates of either laminaran,6 an algal β-(1→3)-glucan, or
linear synthetic oligoglucoside fragments7–11 with carrier proteins were
proved to be immunogenic and protective in mice against infections
induced by Candida albicans and Aspergillus fumigatus.
Important biological properties of β-(1→3)-glucans stimulated con-
siderable activity toward the synthesis of β-(1→3)-oligoglucosides, linear
or containing sporadic β-(1→6)-branchings, of the strictly defined struc-
ture to reveal immunodominant fragments of the polysaccharides or
those responsible for binding to receptors. Several syntheses of
linear8,9,12–20 and branched17,18,21–23 oligomers having different anomeric
functionalization of the monosaccharide at the reducing end have been
published (for review, see Ref. 24).
biomolecular systems25 as model compounds to study biological ac-
tivities of β-(1→3)-glucans.
2. Results and discussion
A common feature of most published syntheses of oligosaccha-
rides related to β-(1→3)-glucans was the use of glycosyl acceptors having
a single free OH group at C-3.8,9,12–16,19–23 If both adjacent hydroxyl groups
at C-2 and C-4 are protected by acyl groups, such glycosyl acceptors
may have low reactivity12 and, more critical, gave sometimes
α-glycosides even upon glycosylation with glycosyl donors bearing a
participating acyl group at O-2.24–27 Therefore, glycosyl acceptors pro-
tected with 4,6-O-benzylidene and 2-O-acyl groups became more
common for the synthesis of such oligosaccharides.8,9,12–16,19–21,28 Another
approach was based on regioselective 3-O-glycosylation of 4,6-O-
benzylidene-protected glycosyl acceptors with a free 2-OH group.17,18
The absence of an acyl protecting group at O-2 would minimize steric
and electronic hindrances to 3-O-glycosylation that enables an effi-
cient chain elongation. This is the approach that allowed the preparation
of the longest synthetic β-(1→3)-oligoglucosides.17,18
The aim of this work was the synthesis of a series of linear
β-(1→3)-oligoglucosides comprising 3–13 monosaccharide units as
Elongation of the oligosaccharide chain was carried out start-
ing from the reducing end by attachment of mono-14,19 or
oligosaccharide8,9,12–18,20 donor blocks followed by removal of a tem-
porary protecting group at O-3. Reiteration of this glycosylation-
deprotection sequence afforded a series of β-(1→3)-oligoglucosides
differing in the length by one, two or more monosaccharide residues.
*
Corresponding author. Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47,
119991 Moscow, Russia. Tel.: +7 499 135 8784; fax: +7 499 135 8784.
E-mail address: nen@ioc.ac.ru (N.E. Nifantiev).
http://dx.doi.org/10.1016/j.carres.2015.10.012
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

2
D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
Scheme 1. Synthesis of disaccharide glycosyl donors 9 and 11. Reagents and conditions: (a) chloroacetic anhydride, 2,4,6-collidine, CH2Cl2, rt; (b) H2NNH2·AcOH, DMF, rt;
(c) Cl3CCN, Cs2CO3, CH2Cl2, rt; (d) TMSOTf, CH2Cl2, −30 °C; (e) BzCl, pyridine, rt; (f) H2NNH2·H2O, CH2Cl2—MeOH, rt.
In this work, we used regioselective 3-O-glycosylation of 4,6-
O-benzylidene-protected 2,3-diol glycosyl acceptors by a disaccharide
donor block for the chain elongation (Scheme 1). Chloroacetyl groups
were applied as temporary protections of the hydroxyl groups at
positions 2 and 3 of the glycosyl donor block.
apply donor 9 for elongation of the oligoglucoside chain (Scheme 2).
NIS-TfOH-promoted coupling of N-Z-protected 3-aminopropyl glu-
coside 1238 with disaccharide 9 displayed almost the same
regioselectivity as the coupling 4 + 6 and resulted in the formation
of necessary (1→3)-linked product 13 (54%) and its positional isomer
14 (10%); subsequent benzoylation of 13 afforded dibenzoate 15. A
set of reagents and conditions for selective removal of the acetyl
groups from 15 was examined; some of them (diluted MeONa in
MeOH, DBU in benzene,36 Et3N in MeOH) exhibited poor
chemoselectivity, others (magnesium methoxide33 or hydrazine
acetate37 in MeOH) demonstrated too slow conversion of starting 15.
We found that a brief treatment of 15 with a large excess of hy-
drazine hydrate (~50 equiv.)a in a mixture CH2Cl2—MeOH provided
fast and selective removal of the acetyl groups in 15 and gave diol
16 in total yield of 79% after repeated treatment of recovered 15.
Although the yield of the conversion 15 → 16 was good enough, one
could assume that accumulation of the 2-O-benzoyl groups upon
elongation of the oligosaccharide chain would lead to decrease of
chemoselectivity of deacetylation. To ensure a reliable transforma-
tion of oligosaccharides longer than the trisaccharide into
corresponding glycosyl acceptors, we decided to revert to the use
of chloroacetyl groups as the temporary protections of the
2,3-diol. To this aim, diacetate 9 was treated with hydrazine hydrate
as described above to give diol 10 (Scheme 1) quantitatively after
two runs of deacetylation; noticeably higher yield of this transfor-
mation as compared to deacetylation of 15, containing two benzoyl
groups, confirmed the assumption of lowering the chemoselectivity
Disaccharide thioglycoside donors 9 and 11 were synthesized as
shown in Scheme 1. First, an attempt to prepare a 2′,3′-di-O-
chloroacetylated donor in a straightforward manner was undertaken.
4,6-O-Benzylidene-D-glucose was subjected to chloroacetylation and
the anomeric chloroacetyl group in obtained tris(chloroacetate) 1 was
selectively removed to afford hemiacetal 2. The latter was converted
into imidate 3; however, its coupling with diol 429 provided orthoester
5 instead of the expected glycoside. The chemical shifts of the signals
for H-1 (δ 5.88) and H-2 (δ 4.44) and the coupling constants pattern
(J1,2 = 5.6 Hz, J2,3 = 3.8 Hz) of the chloroacetylated glucose residue in the
1H NMR spectrum of 5, as well as the position of the signal for C-1 (δ
98.5) and the presence of the signal for quaternary orthoester carbon
(δ 118.1) in the 13C NMR spectrum were in good agreement with the
orthoester structure.30,31 The presence of a correlation peak between
H-2 and the HO-proton of the thioglycoside residue in the COSY spec-
trum indicated that O-3 is involved in the orthoester formation.
Apparently, the electron-withdrawing chloromethyl group at C-2
of the dioxolane ring in 5 stabilizes the orthoester structure and pre-
vents its isomerization to the corresponding glycoside. To overcome
this problem, known 2,3-di-O-acetyl analog 632 was examined in
glycosylation of 4; as a result, (1→3)-linked disaccharide 7 was ob-
tained in acceptable yield (55%) along with (1→2)-regioisomer 8
(8%). The remaining 2-OH group in 7 was benzoylated to give di-
saccharide donor 9. The low-field shift of the signal for H-2 (δ 3.55
→ 5.32) in the 1H NMR spectra upon transformation 7 → 9 indi-
cated the presence of the (1→3)-glycoside bond.
a
The best selectivity was achieved when a ratio substrate–reagent of ~1:50 and
a reaction time of 25 min was employed. In a range of 1–15 equiv. of hydrazine
hydrate, incomplete conversion (<50%) of 15 was observed, while an extension of
reaction time within that range resulted in partial removal of the benzoyl group.
As several methods of selective removal of acetyl groups in the
presence of benzoyl ones are known,33–37 we attempted initially to

D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
3
Scheme 2. Synthesis of trisaccharide acceptor 16. Regents and conditions: (a) NIS, TfOH, mol. sieve AW-300, CH2Cl2, −30 °C; (b) BzCl, pyridine, rt; (c) H2NNH2·H2O, CH2Cl2—MeOH,
rt.
of deacetylation on accumulation of benzoyl groups in the molecule.
Further reaction of 10 with chloroacetic anhydride in the pres-
ence of 2,4,6-collidine provided target glycosyl donor 11 in 92% yield.
NIS-TfOH-promoted glycosylation of acceptor 16 with 11 pro-
ceeded (1→3)-regiospecifically and provided pentasaccharide 17 in
91% yield (Scheme 3). The difference in the regioselectivity of
glycosylation of thioglycoside 4 and alkyl glycoside 12, on the one
hand, and trisaccharide 16, on the other, clearly indicated that a bulky
sugar substituent at the anomeric position of the glycosyl accep-
tor exerts an effective regiocontrol of glycosylation. Acetylation of
17 followed by removal of the chloroacetyl groups produced
pentasaccharide acceptor 18 in 86% yield. Further iteration of
glycosylation and deprotection steps afforded a series of β-(1→3)-
oligoglucosides 19, 21, 23, and 25 in yields of 80–95% having an odd
number of the monosaccharide units.
It should be noted that some internal glucose residues in
pentasaccharide 17 and longer oligoglucosides had unusual for
β-anomers J1,2 coupling constant values (around 5 Hz). The same
effect was observed by other authors8,14,16 and ascribed to a distor-
4
tion of the normal C1 glucose ring conformation.14,16
Transformation of protected oligomers into free 3-aminopropyl
glycosides 26–31 was achieved by successive acidic removal of the
benzylidene groups, basic deacylation and hydrogenolysis of the
N-benzyloxycarbonyl group (Scheme 4).
The structure of oligomers 26–31 was confirmed by 1H and 13
C
NMR data. The spectra of synthesized compounds were very close
Scheme 3. Synthesis of linear β-(1→3)-oligoglucosides by iterative glycosylation-deprotection. Reagents and conditions: (a) NIS, TfOH, mol. sieve AW-300, CH2Cl2. −30 °C
→ −10 °C; (b) AcCl, 2,4,6-collidine, CH2Cl2, rt; (c) thiourea, 2,4,6-collidine, EtOH—EtOAc (1:1), 80 °C.

4
D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
Scheme 4. Preparation of free 3-aminopropyl oligoglucosides. Regents and conditions: (a) 1 M HCl, CHCl3—MeOH, 40–45 °C; (b) MaONa, MeOH then NaOH, aq MeOH, 40–
45 °C; (c) H2, Pd(OH)2/C, aq MeOH, rt.
Table 1
4. Experimental
1H NMRa data for linear oligosaccharides 26–31 (D2O, 600 MHz, 308 K)
Residueb
H-1 (J1,2
)
H-2
H-3
H-4
H-5
H-6a
H-6b
4.1. General methods
Gm
Gm-1
G2–Gm-2
G1
4.76 (8.1)
4.78 (8.0)
4.78 (8.0)
4.52 (8.1)
3.38
3.57
3.57
3.51
3.53
3.79
3.79
3.77
3.43
3.52
3.52
3.52
3.50
3.52
3.52
3.52
3.93
3.93
3.93
3.93
3.73
3.75
3.75
3.75
NMR spectra were recorded on Bruker AMX-400, Bruker DRX-
500, and Bruker Avance 600 instruments. The spectra of protected
carbohydrate derivatives were measured for solutions in CDCl3 or
acetone-d6, and 1H NMR chemical shifts were referenced to the cor-
responding solvent residual signals (δH 7.27 and 2.05 respectively).
13C chemical shifts were referenced to the central resonance of CDCl3
(δC 77.0) and acetone-d6 (δC 29.9). NMR spectra of free oligosaccha-
rides were measured for solutions in D2O using acetone (δH 2.225,
δC 31.45) as an internal standard. Signal assignment was made using
COSY, TOCSY, and HSQC experiments. Monosaccharide residues in
oligosaccharides are numbered by Arabic numerals starting from
the reducing end. HRMS (ESI) were obtained on a MicrOTOF II
(Bruker Daltonics) instrument. Optical rotations were measured using
a JASCO DIP-360 polarimeter at 18–22 °C in solvents specified.
Melting points were determined using a Koffler apparatus. TLC was
performed on Silica Gel 60 F254 plates (E. Merck), and visualiza-
tion was accomplished using UV light or by charring at ~150 °C with
10% (v/v) H3PO4 in ethanol or Mostain reagent (ceric sulfate (1% w/v)
and ammonium molybdate (2.5% w/v) in 10% (v/v) aqueous H2SO4).
Column chromatography was carried out on Silica gel 60 (40–63 μm,
E. Merck). Gel-permeation chromatography of free oligosaccha-
rides was carried out on a TSK HW-40(S) column (2 × 80 cm) in 0.1 M
AcOH. Refractive Index Detector K-2401 (Knauer) was used to
monitor gel-permeation chromatography. All air- or moisture-
sensitive reactions were carried out using dry solvents under dry
argon. Chemicals were purchased from Acros, Fluka, or Aldrich and
used without further purification.
c,d
a
Signals for the aglycon of compounds 26–31 (δ): 4.05, 3.83 (2 m, 2H
OCH2CH2CH2NH2), 3.17 (t, 2H, J = 7.0 Hz, OCH2CH2CH2NH2) 2.02 (m, 2H,
OCH2CH2CH2NH2).
b
Glucose units in oligomers are numbered starting from the reducing end; m is
the total number of monosaccharides in an oligoglucoside.
c
The signals of all internal glucose residues.
These residues are absent in the β-(1→3)-triglucoside 26.
d
Table 2
13C NMRa data for linear oligosaccharides 26–31 (D2O, 150 MHz, 308 K)
Residueb
C-1
C-2
C-3
C-4
C-5
C-6
Gm
104.2
104.0
104.0
103.4
74.9
74.7
74.7
74.2
77.1
85.8
85.7
85.9
71.0
69.6
69.6
69.6
77.5
77.0
77.0
77.0
62.2
62.2
62.2
62.2
Gm-1
G2–Gm-2
G1
a
Signals for the aglycon of compounds 26–31 (δ): 69.3 (OCH2CH2CH2NH2), 39.0
(OCH2CH2CH2NH2); 28.1 (OCH2CH2CH2NH2).
b
See footnotes b–d to Table 1.
to each other in terms of chemical shifts of signals and coupling con-
stant values and differed in the relative intensity of signals belonging
to the internal and terminal monosaccharides. Generalized 1H and
13C NMR data are given in Tables 1 and 2. Coupling constant values
J1,2 of signals for H-1 and chemical shifts of signals for C-1 in 1H and
13C NMR spectra of 26–31 clearly indicated that all glucose units
had β-configuration. Low-field position of all signals for C-3 (except
those of monosaccharides at the nonreducing end) showed all
glucose units to be glycosylated at the position 3 (for more de-
tailed discussion of the NMR data of oligomers 26–31 with regard
to their conformational analysis see Ref. 39).
4.2. 4,6-O-Benzylidene-1,2,3-tri-O-chloroacetyl-D-glucopyranose (1)
Chloroacetic anhydride (578 mg, 3.38 mmol) was added to a
stirred solution of 4,6-O-benzylidene-D-glucopyranose (200 mg,
0.75 mmol) and 2,4,6-collidine (0.60 mL, 4.50 mmol) in dry CH2Cl2
(5 mL). The mixture was stirred at rt for 3 h, quenched with meth-
anol (0.5 mL) and stirring was continued for additional 30 min. The
mixture was diluted with CH2Cl2, washed with aq 1 M HCl, water,
satd aq NaHCO3, dried and concentrated to give crude 1 (375 mg,
100%), which was used in the next step without purification. A small
portion of crude 1 was purified by column chromatography (95:5
toluene–EtOAc) to provide pure 1 as a semicrystalline mixture of
α- and β-anomers in a ratio of ~2:3. 1H NMR (CDCl3, 400 MHz): δ
3.71 (m, 0.6H, H-5β), 3.75–3.83 (m, 2H, H-4αβ, H-6aαβ), 4.01, 4.02,
4.04, 4.09, 4.17 (5 s, 6H, 3 ClCH2α, 3 ClCH2β), 4.06 (m, 0.4H, H-5α),
4.34 (dd, 0.4H, J6b,5 = 4.9 Hz, J6b,6a = 10.4 Hz, H-6bα), 4.41 (dd, 0.6H,
J6b,5 = 4.6 Hz, J6b,6a = 10.2 Hz, H-6bβ), 5.21–5.26 (m, 1H, H-2α, H-2β),
5.45 (t, 0.6H, J = 9.4 Hz, H-3β), 5.51 (s, 0.6H, PhCHβ), 5.53 (s, 0.4H,
3. Conclusion
In conclusion, a series of 3-aminopropyl glycosides of linear
β-(1→3)-oligoglucosides comprising 3–13 glucose residues have been
efficiently synthesized by highly regioselective glycosylation of 2,3-
diol glycosyl acceptors. This representative set of oligoglucosides
will allow examination of the effect of the oligosaccharide length
on the protective properties of corresponding conjugates with a
carrier protein against fungal infections as well as on the effective-
ness of binding to β-(1→3)-glucan receptors.

D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
5
PhCHα), 5.65 (t, 0.4 H, J = 9.9 Hz, H-3α), 5.88 (d, 0.6H, J1,2 = 8.1 Hz,
H-1β), 6.41 (d, 0.4H, J1,2 = 3.8 Hz, H-1α), 7.32–7.46 (m, 5H, Ph). 13C
NMR (CDCl3, 100 MHz): δ 40.2, 40.3, 40.5 (3 ClCH2), 65.4 (C-5α), 67.3
(C-5β), 68.2 (C-6β), 68.3 (C-6α), 70.3 (C-3α), 71.1 (C-2α), 72.4 (C-
2β), 73.1 (C-3β), 77.6 (C-4β), 78.0 (C-4α), 91.0 (C-1α), 93.0 (C-1β),
101.9 (PhCHαβ), 126.2, 128.4, 129.5, 136.4 (Ph), 165.5, 165.8, 166.3,
166.5, 166.7 (ClCH2CO). HRMS (ESI): calcd. for C19H19Cl3O9 [M + Na]+
518.9987; found 518.9975.
the reaction mixture was neutralized with triethylamine, filtered
through a pad of Celite and concentrated. The residue was puri-
fied by silica gel column chromatography (1:1 hexane–EtOAc) to give
orthoester 5 (85 mg, 67%) as a syrup. 1H NMR (CDCl3, 600 MHz): δ
1.35 (t, 3H, J = 7.4 Hz, CH3CH2S), 2.55 (s, 1H, OH), 2.79 (m, 2H,
CH3CH2S), 3.49 (t, 1H, J = 9.6 Hz, H-21), 3.52 (m, 1H, H-51), 3.58 (t,
1H, J = 9.4 Hz, H-41), 3.66 (t, 1H, J = 9.5 Hz, H-42), 3.67 (t, 1H,
J = 10.8 Hz, H-61a), 3.76 (t, 1H, J = 10.2 Hz, H-62a), 3.88 and 3.95 (2
d, each 1H, Jgem = 12.3 Hz, ClCH2), 4.03–4.10 (m, 3H, H-31, ClCH2), 4.14
(m, 1H, H-52), 4.37 (dd, 1H, J6b,5 = 4.7 Hz, J6b,6a = 10.5 Hz, H-62b), 4.40
(dd, 1H, J6b,5 = 5.2 Hz, J6b,6a = 10.5 Hz, H-61b), 4.43–4.47 (m, 2H, H-11,
H-22), 5.50, 5.51 (2 s, 2H, 2 PhCH), 5.54 (dd, 1H, J2,3 = 3.8 Hz,
J3,4 = 9.1 Hz, H-32), 5.88 (d, 1H, J1,2 = 5.6 Hz, H-12), 7.35–7.50 (m, 10H,
2 Ph). 13C NMR (CDCl3, 150 MHz): δ 15.4 (CH3CH2S), 24.8 (CH3CH2S),
40.7, 44.1 (2 ClCH2), 62.6 (C-5′), 68.5 (C-6′), 68.6 (C-6), 71.0 (C-5),
72.5 (C-2), 75.0 (C-3′), 75.5 (C-2′), 76.5 (C-4′), 76.8 (C-3), 79.5 (C-
4), 87.3 (C-1), 98.5 (C-1′), 101.5, 102.0 (2 PhCH), 118.1 (orthoester
C), 126.1–136.8 (2 Ph), 166.1 (ClCH2CO). HRMS (ESI): calcd. for
C32H36Cl2O12S [M + Na]+ 737.1197; found 737.1188.
4.3. 4,6-O-Benzylidene-2,3-di-O-chloroacetyl-D-glucopyranose (2)
Hydrazine acetate (48 mg, 0.52 mmol) was added to a solution
of 1 (200 mg, 0.40 mmol) in anhydrous DMF (5 mL). The mixture
was stirred at rt for 1 h, diluted with EtOAc, washed with aq 1 M
HCl, water, satd aq NaHCO3, dried and concentrated. The residue was
purified by silica gel column chromatography (85:15 toluene–
EtOAc) to give syrupy hemiacetal 2 (152 mg, 90%) as a mixture of
α- and β-anomers in a ratio of about 5:3. 1H NMR (CDCl3, 400 MHz):
δ 2.90 (d, 1H, J1,OH = 3.7 Hz, OHα), 3.26 (d, 0.6H, J1,OH = 8.0 Hz, OHβ),
3.52 (m, 0.6H, H-5β), 3.62–3.77 (m, 3.2H, H-4αβ, H-6aαβ), 3.97, 4.02,
4.03 (3 s, 6.4H, 2 ClCH2α, 2 ClCH2β), 4.13 (m, 1H, H-5α), 4,24 (dd,
1H, J6b,5 = 5.0 Hz, J6b,6a = 10.2 Hz, H-6bα), 4.32 (dd, 0.6H, J6b,5 = 4.9 Hz,
J6b,6a = 10.5 Hz, H-6bβ), 4.82 (t, 0.6H, J = 7.9 Hz, H-1β), 4.90–4.96 (m,
1.6H, H-2αβ), 5.37 (t, 0.6H, J = 9.5 Hz, H-3β), 5.43–5.45 (m, 2.6H, H-1α,
PhCHαβ), 5.64 (t, 1H, J = 9.8 Hz, H-3α), 7.28–7.39 (m, 8H, Ph). 13C NMR
(CDCl3, 100 MHz): δ 40.5, 40.6, 40.8 (2 ClCH2), 62.5 (C-5α), 66.7 (C-
5β), 68.5 (C-6β), 68.8 (C-6α), 70.6 (C-3α), 72.9 (C-3β), 73.2 (C-2α),
75.3 (C-2β), 78.2 (C-4β), 78.8 (C-4α), 90.8 (C-1α), 95.6 (C-1β), 101.8
(PhCHαβ), 126.2, 128.4, 129.3, 136.5, 136.7 (Ph), 166.6, 166.8, 167.0,
167.3 (ClCH2CO). HRMS (ESI): calcd. for C17H18Cl2O8 [M + Na]+
443.0271; found 443.0268.
4.6. Ethyl 2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-
(1→3)-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (7) and ethyl
2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→2)-4,6-
O-benzylidene-1-thio-β-D-glucopyranoside (8)
Trimethylsilyl triflate (50 μL, 0.26 mmol) was added to a stirred
solution of imidate 6 (4.30 g, 8.66 mmol) and diol 4 (4.20 g,
13.46 mmol) in dichloromethane (70 mL) at −30 °C, the resulting
mixture was stirred at this temperature for 10 min, and quenched
with triethylamine. The solvent was evaporated and the residue was
subjected to silica gel column chromatography (toluene → 93:7
toluene–acetone) to provide starting diol 4 (1.45 g, 35%) and disac-
charides 7 (3.07 g, 55%), and 8 (446 mg, 8%).
4.4. 4,6-O-Benzylidene-2,3-di-O-chloroacetyl-D-glucopyranosyl
trichloroacetimidate (3)
Compound 7, crystalline solid, mp 214–215 °C (EtOAc–hexane),
[α]D −84 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz): δ 1.31 (t, 3H,
J = 7.3 Hz, CH3CH2S), 2.04, 2.05 (2 s, each 3H, 2 CH3CO), 2.68 (br. s,
1H, OH), 2.75 (m, 2H, CH3CH2S), 3.43 (m, 1H, H-52), 3.49 (m, 1H, H-51),
3.43 (br. t, 1H, J = 9.1 Hz, H-21), 3.65 (t, 1H, J = 9.7 Hz, H-41), 3.70 (t,
1H, J = 10.3 Hz, H-62a), 3.74 (t, 1H, J = 10.3 Hz, H-42), 3.77 (t, 1H,
J = 10.4 Hz, H-61a), 3.83 (t, 1H, J = 8.9 Hz, H-31), 4.12 (dd, 1H,
J6b,5 = 5.0 Hz, J6b,6a = 10.5 Hz, H-62b), 4.35 (dd, 1H, J6b,5 = 4.9 Hz,
J6b,6a = 10.5 Hz, H-61b), 4.45 (d, 1H, J1,2 = 7.5 Hz, H-11), 4.94 (d, 1H,
J1,2 = 7.5 Hz, H-12), 5.05 (t, 1H, J = 8.1 Hz, H-22), 5.28 (t, 1H, J = 9.1 Hz,
H-32), 5.35, 5.55 (2 s, each 1H, 2 PhCH), 7.30–7.50 (m, 10H, 2 Ph).
13C NMR (CDCl3, 150 MHz): δ 15.2 (CH3CH2S), 20.7, 20.8 (2 CH3CO),
24.6 (CH3CH2S), 66.3 (C-5′), 68.4 (C-6′), 68.5 (C-6), 70.8 (C-5), 71.8
(C-3′), 72.5 (C-2), 73.0 (C-2′), 78.0 (C-4′), 79.4 (C-4), 82.3 (C-3), 86.5
(C-1), 101.3, 101.4 (2 PhCH), 101.7 (C-1′), 125.9–137.0 (2 Ph), 170.0,
170.1 (2 CH3CO). HRMS (ESI): calcd. for C32H38O12S [M + Na]+ 669.1976;
found 669.1965.
Compound 8, crystalline solid, mp 172–174 °C (EtOAc–hexane),
[α]D −91 (c 1, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.21 (t, 3H,
J = 7.3 Hz, CH3CH2S), 1.96, 1.99 (2 s, each 3H, 2 CH3CO), 2.59–2.72 (m,
3H, CH3CH2S, ОH), 3.36 (m, 1 H, H-51), 3.43 (t, 1H, J = 9.3 Hz, H-41),
3.47 (m, 1H, H-52), 3.62 (t, 1H, J = 8.5 Hz, H-21), 3.65 (t, 1H, J = 10.2 Hz,
H-62a), 3.71 (1 H, t, J = 9.7 Hz, H-42), 3.74–3.82 (m, 2H, H-31, H-61a),
4.22–4.32 (m, 2H, H-61b, 62b), 4.42 (d, 1H, J1,2 = 9.6 Hz, H-11), 4.97
(dd, 1H, J2,3 = 8.8 Hz, H-22), 5.07 (d, 1H, J1,2 = 7.8 Hz, H-12), 5.25 (t,
1H, J = 9.3 Hz, H-32), 5.42, 5.45 (2 s, each 1H, 2 PhCH), 7.25–7.42 (m,
10H, 2 Ph). 13C NMR (CDCl3, 100 MHz): δ 14.8 (CH3CH2S), 20.8, 20.9
(2 CH3CO), 24.5 (CH3CH2S), 66.4 (C-52), 68.6 (C-62), 68.7 (C-61), 70.1
(C-51), 72.1 (C-32), 73.0 (C-22), 75.4 (C-31), 78.2 (C-42), 79.9 (C-21),
80.4 (C-41), 84.1 (C-11), 101.1 (C-12), 101.6, 102.0 (2 PhCH),
126.1–136.9 (2 Ph), 169.9, 170.2 (2 CH3CO). HRMS (ESI): calcd. for
C32H38O12S [M + Na]+ 669.1976; found 669.1971.
Trichloroacetonitrile (0.5 mL) and Cs2CO3 (50 mg) were added
to a solution of 2 (152 mg, 0.36 mmol) in dichloromethane (5 mL),
the mixture was stirred at rt for 30 min, diluted with toluene,
and filtered through a pad of silica gel (toluene → 95:5 toluene–
EtOAc) to give trichloroacetimidate 3 (187 mg, 92%) as a syrupy
anomeric mixture; α:β-ratio ~1:1. 1H NMR (C6D6, 600 MHz): δ
3.05 (m, 1H, H-5β), 3.19–3.52 (m, 12H, H-4αβ, H-6aαβ, 2 ClCH2αβ),
3.95 (dd, 1H, J6b,5 = 4.9 Hz, J6b,6a = 10.3 Hz, H-6bβ), 4.05 (dd, 1H,
J6b,5 = 5.0 Hz, J6b,6a = 10.5 Hz, H-6bα), 4.22 (m, 1H, H-5α), 5.07 (s,
1H, PhCH), 5.19 (dd, 1H, J2,3 = 9.9 Hz, H-2α), 5.22 (s, 1H, PhCH),
5.39 (t, 1H, J = 9.8 Hz, H-3β), 5.42 (t, 1H, J = 9.1 Hz, H-2β), 5.87 (d,
1H, J1,2 = 7.6 Hz, H-1β), 5.98 (t, 1H, J = 9.9 Hz, H-3α), 6.70 (d, 1H,
J1,2 = 3.7 Hz, H-1α), 7.00–7.57 (m, 10H, Phαβ), 8.42, 8.59 (2 s, each
1H, NHαβ). 13C NMR (C6D6, 150 MHz): δ 40.3, 40.4, 40.5, 40.6 (2
ClCH2), 66.0 (C-5α), 67.2 (C-5β), 68.5 (C-6β), 68.6 (C-6α), 70.9
(C-3α), 72.3 (C-2α), 73.0 (C-2β), 73.5 (C-3β), 77.9 (C-4β), 78.4
(C-4α), 93.9 (C-1α), 96.0 (C-1β), 102.2, 102.5 (PhCHαβ), 127.0,
128.2, 128.4, 129.7, 129.8, 137.5, 137.6 (Ph), 161.2, 161.4 (C = NH),
166.4, 166.7, 166.8, 167.0 (ClCH2CO). HRMS (ESI): calcd. for
C19H18Cl5NO8 [M + Na]+ 585.9367; found 585.9364.
4.5. 4,6-O-Benzylidene-3-O-chloroacetyl-1,2-O-[1-(ethyl 4,6-O-
benzylidene-1-thio-β-D-glucopyranoside-3-O-yl)-2-
chloroethylidene]-α-D-glucopyranose (5)
A mixture of thioglycoside 4 (61 mg, 0.194 mmol, 1.1 eq.),
trichloroacetimidate 3 (100 mg, 0.177 mmol) and MS 4 Å (150 mg)
in dry CH2Cl2 (2 mL) was stirred at rt for 30 min. Then the mixture
was cooled to −30 °C, and a solution of TMSOTf in dry CH2Cl2 (0.1 mL,
prepared by dissolving 34 μL of TMSOTf in 1 mL of dry CH2Cl2,
0.018 mmol) was added. After being stirred for 30 min at −30 °C,

6
D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
4.7. Ethyl 2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-
(1→3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (9)
(t, 1H, J = 10.3 Hz, H-61a), 3.99 (s, 2H, ClCH2), 4.01 (d, 1H, J = 14.9 Hz,
ClCHbHa), 4.22 (t, 1H, J = 9.1 Hz, H-31), 4.30 (dd, 1H, J6b,5 = 4.9 Hz,
J6b,6a = 10.5 Hz, H-62b), 4.42 (dd, 1H, J6b,5 = 4.8 Hz, J6b,6a = 10.5 Hz, H-61b),
4.64 (d, 1H, J1,2 = 10.1 Hz, H-11), 4.78 (d, 1H, J1,2 = 7.6 Hz, H-12), 5.09 (t,
1H, J = 8.4 Hz, H-22), 5.16 (t, 1H, J = 9.3 Hz, H-32), 5.35 (t, 1H, J = 9.3 Hz,
Benzoyl chloride (1.2 mL, 10.3 mmol) and DMAP (100 mg) were
added to a solution of 7 (3.00 g, 4.64 mmol) in pyridine (20 mL) and
the mixture was stirred for 20 h at rt. Water (1 mL) was added and
stirring was continued for 30 min. The mixture was diluted with
CH2Cl2, washed successively with aq 1 M HCl, water, and satd aq
NaHCO3, dried and concentrated. The residue was crystallized from
ethanol to give 9 (3.20 g, 92%) as white crystals, mp 207–208 °C, [α]D
−69 (c 1, CHCl3). 1H NMR (CDCl3, 500 MHz): 1.20 (t, 3H, J = 7.5 Hz,
CH3CH2S), 1.73, 1.94 (2 s, each 3H, 2 CH3CO), 2.70 (m, 2H, CH3CH2S),
3.37 (m, 1H, H-52), 3.55 (m, 1H, H-51), 3.64 (t, 1H, J = 9.5 Hz, H-42),
3.66 (t, 1H, J = 10.0 Hz, H-62a), 3.74 (t, 1H, J = 9.4 Hz, H-41), 3.80 (t,
1H, J = 10.3 Hz, H-61a), 4.16 (t, 1H, J = 9.1 Hz, H-31), 4.20 (dd, 1H,
J6b,5 = 5.0 Hz, J6b,6a = 10.5 Hz, H-62b), 4.38 (dd, 1H, J6b,5 = 4.9 Hz,
J6b,6a = 10.5 Hz, H-61b), 4.59 (d, 1H, J1,2 = 10.0 Hz, H-11), 4.72 (d, 1H,
J1,2 = 7.6 Hz, H-12), 4.96 (t, 1H, J = 8.8 Hz, H-22), 5.04 (t, 1H, J = 9.2 Hz,
H-32), 5.32 (t, 1H, J = 9.4 Hz, H-21), 5.35, 5.57 (2 s, each 1H, 2 PhCH),
7.30–8.06 (m, 15H, 3 Ph). 13C NMR (CDCl3, 125 MHz): δ 14.7
(CH3CH2S), 20.1, 20.7 (2 CH3CO), 23.4 (CH3CH2S), 66.1 (C-52), 68.5 (2C,
C-61, 62), 71.1 (C-51), 71.9, 71.95, 72.0 (C-21, 22, 32), 78.1 (C-42), 78.8
(C-41), 79.7 (C-31), 84.3 (C-11), 100.9 (C-12), 101.1, 101.3 (2 PhCH),
126.0–137.1 (3 Ph), 164.9 (PhCO), 169.6, 170.0 (2 CH3CO). HRMS (ESI):
calcd. for C39H42O13S [M + Na]+ 773.2238; found 773.2216.
H-21), 5.42, 5.61 (2 s, each 1H, 2 PhCH), 7.33–8.11 (m, 15H, 3 Ph). 13
C
NMR (CDCl3, 150 MHz): δ 14.7 (CH3CH2S), 24.0 (CH3CH2S), 40.2, 40.4
(2 ClCH2), 66.2 (C-52), 68.4 (C-62), 68.5 (C-61), 71.1 (C-51), 72.2 (C-21),
73.2, (C-22), 73.3 (C-32), 77.9 (C-41), 78.6 (C-42), 79.8 (C-31), 84.2 (C-
11), 100.5 (C-12), 101.1, 101.5 (2 PhCH), 126.0–137.1 (3 Ph), 165.2 (PhCO),
166.5, 166.6 (2 ClCH2CO). HRMS (ESI): calcd. for C39H40Cl2O13S [M + Na]+
841.1459; found 841.1459.
4.10. 3-Benzyloxycarbonylaminopropyl
2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-
benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-
benzylidene-β-D-glucopyranoside (13) and
3-benzyloxycarbonylaminopropyl 2,3-di-O-acetyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→2)-4,6-O-benzylidene-β-D-
glucopyranoside (14)
A mixture of diol 12 (1.16 g, 2.53 mmol), thioglycoside 9 (1.88 g,
2.50 mmol) and mol. sieve AW-300 (3.7 g) in CH2Cl2 (40 mL) was
stirred for 30 min at rt and then cooled to −30 °C. NIS (650 mg,
2.89 mmol) was added, stirring was continued for 30 min at −30 °C,
and a solution of TfOH (50 μL, 0.56 mmol) in CH2Cl2 (1 mL) was
added. The mixture was stirred for additional 30 min, quenched by
adding satd aq NaHCO3 and aq 1 M Na2S2O3 and filtered through a
layer of Celite. The solids were washed with CH2Cl2, the combined
filtrates were allowed to separate, and the organic layer was dried
and concentrated. Column chromatography of the residue (toluene
→ 95:5 toluene–EtOAc) provided (1→3)-trisaccharide 13 (1.56 g, 54%),
and (1→2)-trisaccharide 14 (287 mg, 10%).
4.8. Ethyl 4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-
benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (10)
Hydrazine hydrate (4.7 mL, 97 mmol) was added to a solution
of 9 (1.42 g, 1.89 mmol) in CH2Cl2 (14 mL) and MeOH (28 mL). The
mixture was stirred for 25 min, diluted with water (150 mL), and
extracted with CH2Cl2 (3 × 50 mL). The combined extracts were
washed with brine, dried, and concentrated. Column chromatog-
raphy of the residue (CH2Cl2 → 95:5 CH2Cl2–acetone) afforded starting
9 (120 mg, 8.5%) and diol 10 (1.15 g, 91.5%). Treatment of recov-
ered 9 as described above provided more 10 (110 mg); total yield
of diol 10 made up 1.26 g (100%), amorphous solid, [α]D −54 (c 1,
CHCl3). 1H NMR (CDCl3, 600 MHz): δ 1.26 (t, 3H, J = 7.4 Hz, CH3CH2S),
2.53 (s, 1H, OH-32), 2.76 (m, 2H, CH3CH2S), 2.90 (d, 1H, JOH,2 = 2.4 Hz,
OH-22), 3.30 (m, 1H, H-52), 3.44–3.48 (m, 3H, H-22, 42, 62a), 3.58–3.65
(m, 2H, H-32, 51), 3.81 (t, 1H, J = 9.6 Hz, H-41), 3.83–3.87 (m, 2H, H-61a,
62b), 4.21 (t, 1H, J = 9.0 Hz, H-31), 4.43 (dd, 1H, J6b,5 = 5.1 Hz,
J6b,6a = 10.5 Hz, H-61b), 4.47 (d, 1H, J1,2 = 7.8 Hz, H-12), 4.73 (d, 1H,
J1,2 = 10.2 Hz, H-11), 5.37 (t, 1H, J = 9.3 Hz, H-21), 5.43, 5.63 (2 s, each
1H, 2 PhCH), 7.32–8.08 (m, 15H, 3 Ph). 13C NMR (CDCl3, 150 MHz):
δ 14.8 (CH3CH2S), 24.1 (CH3CH2S), 66.6 (C-52), 68.4 (C-62), 68.5 (C-
61), 70.9 (C-51), 71.9 (C-21), 72.8 (C-32), 73.4 (C-22), 79.1 (C-41), 79.3
(C-31), 80.2 (C-42), 84.3 (C-11), 101.6, 101.8 (2 PhCH), 103.0 (C-12),
(1→3)-Trisaccharide 13: amorphous solid, [α]D −34 (c 1, CHCl3).
1H NMR (CDCl3, 600 MHz): δ 1.75 (m, 2H, NHCH2CH2CH2O),
1.83, 2.00 (2 s, each 3H, CH3CO), 3.22 (m, 1H, NHCHaHbCH2CH2O),
3.39–3.48 (m, 4H, NHCHaHbCH2CH2O, H-21, 51, 53), 3.53 (m, 1H,
NHCH2CH2CHaHbO), 3.58 (m, 1H, H-52), 3.61 (t, 1H, J = 9.4 Hz, H-41),
3.70 (t, 2H, J = 10.0 Hz, H-43, 63a), 3.73 (t, 1H, J = 10.6 Hz, H-62a), 3.77
(t, 1H, J = 10.4 Hz, H-61a), 3.85 (t, 1H, J = 9.0 Hz, H-31), 3.92 (m, 1H,
NHCH2CH2CHaHbO), 3.96 (t, 1H, J = 9.4 Hz, H-42), 4.13 (t, 1H, J = 7.9 Hz,
H-32), 4.21 (dd, 1H, J6b,5 = 4.6 Hz, J6b,6a = 10.3 Hz, H-62b), 4.24 (dd, 1H,
J6b,5 = 5.0 Hz, J6b,6a = 10.5 Hz, H-63b), 4.28 (d, 1H, J1,2 = 7.7 Hz, H-11),
4.33 (dd, 1H, J6b,5 = 4.8 Hz, J6b,6a = 10.5 Hz, H-62b), 4.86 (d, 1H,
J1,2 = 7.5 Hz, H-13), 5.02 (t, 1H, J = 9.0 Hz, H-23), 5.04 (br. t, 1H, NH),
5.11 (s, 2H, PhCH2), 5.16 (t, 1H, J = 9.2 Hz, H-33), 5.17 (d, 1H, J1,2 = 6.1 Hz,
H-12), 5.27, 5.40, 5.56 (3 s, each 1H, 3 PhCH), 5.31 (t, 1H, J = 6.7 Hz,
H-22), 7.30–8.09 (m, 25H, 5 Ph). 13C NMR (CDCl3, 150 MHz): δ 20.5,
20.6 (2 CH3CO), 29.4 (NHCH2CH2CH2O), 38.1 (NHCH2CH2CH2O), 66.1
(C-52), 66.2 (C-53), 66.5 (C-51), 66.8 (PhCH2), 67.3 (NHCH2CH2CH2O),
68.5, 68.6, 68.8 (C-61, 62, 63), 72.1 (C-33), 72.2 (C-23), 74.4 (C-22), 74.5
(C-21), 78.2 (C-43), 78.3 (C-42), 78.7 (C-32), 79.2 (C-41), 79.8 (C-31),
100.5 (C-13), 100.9 (2C, C-12, PhCH), 101.4, 101.5 (2 PhCH), 103.2
(C-11), 156.5 (OCONH), 165.3 (PhCO), 169.6, 170.0 (2 CH3CO). HRMS
(ESI): calcd. for C61H65NO21 [M + Na]+ 1170.3941; found 1170.3901.
(1→2)-Trisaccharide 14: amorphous solid, [α]D −38 (c 1, CHCl3).
1H NMR (CDCl3, 600 MHz): δ 1.74 (s, 3H, CH3CO), 1.82 (m, 2H,
NHCH2CH2CH2O), 1.96 (s, 3H, CH3CO), 3.30–3.36 (m, 3H,
NHCH2CH2CH2O, H-51), 3.37–3.44 (m, 2H, H-41, 53), 3.52–3.58 (m,
2H, H-21, 52), 3.63–3.72 (m, 5H, H-31, 43, 61a, 63a, NHCH2CH2CHaHbO),
3.78–3.85 (m, 2H, H-42, 62a), 3.93 (m, 1H, NHCH2CH2CHaHbO), 4.18
(t, 1H, J = 8.8 Hz, H-32), 4.20 (dd, 1H, J6b,5 = 4.8 Hz, J6b,6a = 10.3 Hz,
H-63b), 4.29 (dd, 1H, J6b,5 = 5.0 Hz, J6b,6a = 10.6 Hz, H-61b), 4.33 (dd,
1H, J6b,5 = 4.6 Hz, J6b,6a = 10.2 Hz, H-62b), 4.41 (d, 1H, J1,2 = 7.6 Hz, H-11),
4.77 (d, 1H, J1,2 = 7.5 Hz, H-13), 4.98 (t, 1H, J = 8.4 Hz, H-23), 5.09 (t,
126.1–136.9 (3 Ph), 156.8 (PhCO). HRMS (ESI): calcd. for C35H38O11
S
[M + K]+ 705.1766; found 705.1778.
4.9. Ethyl 4,6-O-benzylidene-2,3-di-O-chloroacetyl-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-
glucopyranoside (11)
Chloroacetic anhydride (845 mg, 4.95 mmol) was added to a solu-
tion of diol 10 (1.10 g, 1.65 mmol) and 2,4,6-collidine (0.88 mL, 6.6 mmol)
in CH2Cl2 (10 mL). The mixture was stirred for 2 h at rt, then MeOH
(1 mL) was added and stirring was continued for next 30 min. After di-
lution with CH2Cl2, the mixture was successively washed with aq 1 M
HCl, water, satd aq NaHCO3, dried and concentrated. The residue was
recrystallized from ethanol to afford 11 (1.24 g, 92%), mp 178–179 °C,
[α]D −57 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz): δ 1.32 (t, 3H, J = 7.3 Hz,
CH3CH2S), 2.74 (m, 2H, CH3CH2S), 3.44 (m, 1H, H-52), 3.60 (m, 1H, H-51),
3.70–3.75 (m, 3H, H-42, 62a, ClCHbHa), 3.78 (t, 1H, J = 9.4 Hz, H-41), 3.84

D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
7
1H, J = 9.3 Hz, H-33), 5.12 (m, 2H H-12, PhCHaHb), 5.21 (d, 1H,
J = 12.3 Hz, PhCHaHb), 5.33 (t, 1H, J = 8.0 Hz, H-22), 5.36, 5.40, 5.44
(3 s, each 1H, 3 PhCH), 5.45 (br. t, 1H, NH), 7.30–8.07 (m, 25H,
5 Ph). 13C NMR (CDCl3, 150 MHz): δ 20.1, 20.7 (2 CH3CO), 28.9
(NHCH2CH2CH2O), 38.1 (NHCH2CH2CH2O), 65.8 (C-51), 66.1 (C-53), 66.5
(2C, C-52, PhCH2), 68.1 (NHCH2CH2CH2O), 68.4 (C-62), 68.5 (2C, C-61,
63), 72.0 (C-33), 72.1 (C-23), 73.7 (C-31), 74.2 (C-22), 78.1 (C-43), 78.4
(C-42), 78.7 (C-32), 80.2 (C-41), 81.1 (C-21), 100.9 (C-13), 101.1, 101.4
(2 PhCH), 101.7 (C-12), 101.9 (2C, C-11, PhCH), 126.0–137.2 (5 Ph),
156.6 (OCONH), 165.3 (PhCO), 169.5, 170.0 (2 CH3CO). HRMS (ESI):
calcd. for C61H65NO21 [M + Na]+ 1170.3941; found 1170.3937.
80.2 (C-43), 100.0 (C-12), 100.9 (C-11), 101.3, 101.7, 101.8 (3 PhCH), 102.5
(C-13), 126.0–137.0 (6 Ph), 156.4 (OCONH), 164.9, 165.3 (2 PhCO). HRMS
(ESI): calcd. for C64H65NO20 [M + Na]+ 1190.3992; found 1190.4002.
4.13. General procedure A. Elongation of the oligosaccharide chain
by glycosylation of diol acceptors with donor 11
A mixture of donor 11 (1.05–1.15 equiv.), a diol acceptor and mol.
sieve AW-300 (1.5 g/mmol) in CH2Cl2 (15 mL/mmol) was stirred at
rt for 30 min and cooled to −30 °C. NIS (1.5 equiv. relative to 11) was
added and the resulting mixture was stirred for additional 30 min
at the same temperature. Then a solution of TfOH (7 molar %) in
CH2Cl2 was added at −30 °C, and stirring was continued for next 2 h,
while the temperature was gradually increased to −10 °C. The re-
action mixture was quenched with satd aq NaHCO3 and aq 1 M
Na2S2O3 and filtered through a Celite layer. The solids were washed
with CH2Cl2, the organic layer from the combined filtrates was sepa-
rated, dried, and concentrated. The residue was purified by silica
gel column chromatography (9:1 toluene–EtOAc → 85:15 toluene–
EtOAc) to provide a glycosylation product.
4.11. 3-Benzyloxycarbonylaminopropyl 2,3-di-O-acetyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranoside (15)
Trisaccharide 13 was benzoylated as described for 9 to provide
trisaccharide 15 (97%), amorphous solid, [α]D −33 (c 1, CHCl3). 1H
NMR (CDCl3, 600 MHz): δ 1.66 (m, 2H, NHCH2CH2CH2O), 1.80, 1.96
(2 s, each 3H, 2 CH3CO), 3.09 (m, 2H, NHCH2CH2CH2O), 3.35 (m, 1H,
H-53), 3.43 (m, 1H, NHCH2CH2CHaHbO), 3.48 (m, 1H, H-52), 3.57 (m,
1H, H-51), 3.62–3.69 (m, 3H, H-43, 62a, 63a), 3.78–3.89 (m, 4H, H-41,
42, 61a, NHCH2CH2CHaHbO), 4.01 (t, 1H, J = 7.5 Hz, H-32), 4.18–4.22
(m, 3H, H-31, 62b, 63b), 4.37 (dd, 1H, J6b,5 = 4.8 Hz, J6b,6a = 10.2 Hz,
H-61b), 4.57 (d, 1H, J1,2 = 7.2 Hz, H-11), 4.72 (d, 1H, J1,2 = 7.8 Hz, H-13),
4.88–4.96 (m, 3H, H-12, 23, NH), 5.04–5.08 (m, 3H, H-33, PhCH2), 5.17
(s, 1H, PhCH), 5.18–5.23 (m, 2H, H-21, 22), 5.37, 5.59 (2 s, each 1H,
2 PhCH), 7.30–7.85 (m, 30 H, 6 Ph). 13C NMR (CDCl3, 150 MHz): δ
20.2, 20.7 (2 CH3CO), 29.4 (NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O),
66.0 (C-52), 66.1 (C-53), 66.4 (PhCH2), 66.5 (C-51), 67.2
(NHCH2CH2CH2O), 68.5, 68.6 (C-62, 63), 68.7 (C-61), 71.9 (2C, C-23,
33), 73.8 (2C, C-22, 23), 77.0 (C-31), 78.1 (C-42), 78.2 (C-43), 78.4 (C-
32), 79.0 (C-41), 99.9 (C-12), 100.3 (C-13), 100.8 (PhCH), 101.2 (C-
11), 101.3, 101.6 (2 PhCH), 126.0–137.2 (6 Ph), 156.4 (OCONH), 164.5,
164.8 (2 PhCO), 169.6, 170.0 (2 CH3CO). HRMS (ESI): calcd. for
C68H69NO22 [M + Na]+ 1274.4203; found 1274.4210.
4.14. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-2,3-di-O-
chloroacetyl-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (17)
Pentasaccharide 17 was prepared by glycosylation of diol 16 with
11 according to the general procedure A in 91% yield, amorphous
powder, [α]D −23 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz, selected data):
δ 1.68 (m, 2H, NHCH2CH2CH2O), 2.90 (br. s, 1H, OH), 3.09, 3.12 (2 m, 2H,
NHCH2CH2CH2O), 3.33 (br. t, J = 8.5 Hz, H-23), 3.99, 4.07 (2 d, each 1H,
J = 15.9 Hz, ClCH2), 4.09 (s, 2H, ClCH2), 4.36 (d, 1H, J1,2 = 8.0 Hz, H-13),
4.61 (d, 1H, J1,2 = 7.2 Hz, H-1), 4.72 (s, 1H, PhCH), 5.00 (d, 1H, J1,2 = 6.9 Hz,
H-1), 5.07 (d, 1H, J1,2 = 4.8 Hz, H-1), 5.08 (s, 2H, PhCH2), 5.09 (d, 1H,
J1,2 = 7.4 Hz, H-1), 5.16 (t, 2H, J = 8.6 Hz, 2 H-2), 5.26 (t, 1H, J = 7.7 Hz, H-2),
5.32 (s, 1H, PhCH), 5.40 (t, 1H, J = 9.5 Hz, H-35), 5.43, 5.48, 5.58 (3 s, each
1H, 3 PhCH), 7.18–8.09 (m, 45H, 9 Ph). 13C NMR (CDCl3, 150 MHz, se-
lected data): δ 29.4 (NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 40.6, 40.8
(2 ClCH2), 66.4 (PhCH2), 98.5 (C-1), 99.2 (C-1), 100.2 (C-1), 100.4 (PhCH),
101.1 (C-1), 101.3, 101.6, 101.7, 101.8 (4 PhCH), 102.0 (C-13), 126.0–137.3
(9 Ph), 156.4 (OCONH), 164.9, 165.0, 165.3, 166.2, 166.7 (3 PhCO, 2
ClCH2CO). HRMS (ESI): calcd. for C101H99Cl2NO33 [M + Na]+ 1946.5369;
found 1946.5369.
4.12. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (16)
Hydrazine hydrate (2.6 mL, 54 mmol) was added to a solution of 15
(1.55 g, 1.24 mmol) in CH2Cl2 (8 mL) and MeOH (16 mL) at rt. After being
stirred for 25 min, the mixture was diluted with water (100 mL) and
extracted with CH2Cl2 (3 × 30 mL). The combined extracts were washed
with brine, dried, and concentrated. Column chromatography of the
residue CH2Cl2 → 85:15 CH2Cl2–acetone gave unreacted 15 (200 mg, 13%)
and diol 16 (1.03 g, 71%). Repeated deacetylation of recovered 15 pro-
vided an additional portion of 16 (110 mg). Total yield of 16 made up
4.15. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-2,3-di-O-
chloroacetyl-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (19)
1
1.14 g (79%), amorphous solid, [α]D −21 (c 1, CHCl3). H NMR (CDCl3,
600 MHz, selected data): δ 1.64 (m, 2H NHCH2CH2CH2O), 3.08, 3.12 (2 m,
2H, NHCH2CH2CH2O), 3.26 (m, 1H, H-53), 3.38–3.52 (m, 4H, H-23, 43, 63a,
NHCH2CH2CHaHbO), 3.48 (m, 1H, H-51), 3.59–3.65 (m, 2H, H-33, H-52),
3.68 (t, 1H, J = 10.2 Hz, H-61a), 3.79–3.88 (m, 3H, H-62a, 63b,
NHCH2CH2CHaHbO), 3.86 (t, 1H, J = 9.1 Hz, H-41), 3.91 (t, 1H, J = 9.2 Hz,
H-42), 4.04 (t, 1H, J = 8.1 Hz, H-31), 4.18–4.22 (m, 2H, H-32, 61b), 4.39 (dd,
1H, J6b,5 = 4.7 Hz, J6b,6a = 10.5 Hz, H-62b), 4.44 (d, 1H, J1,2 = 7.8 Hz,
H-13), 4.62 (d, 1H, J1,2 = 7.0 Hz, H-11), 4.97 (br. t, 1H, NH), 5.03 (d,
1H, J1,2 = 6.6 Hz, H-12), 5.07 (s, 2H, PhCH2), 5.23–5.27 (m, 2H,
H-21, 22), 5.35, 5.41, 5.58 (3 s, each 1H, PhCH), 7.30–8.89 (m, 30H,
6 Ph). 13C NMR (CDCl3, 150 MHz): δ 29.4 (NHCH2CH2CH2O), 38.0
(NHCH2CH2CH2O), 65.9 (C-51), 66.2 (C-52), 66.4 (C-53), 66.6 (PhCH2), 67.2
(NHCH2CH2CH2O), 68.3 (C-63), 68.6 (C-61), 68.8 (C-62), 72.7 (C-33), 73.5
(2C), 73.6 (C-21, 22, 23), 77.9 (C-32), 78.2 (C-31), 78.5 (C-41), 79.3 (C-42),
Heptasaccharide 19 (83%) was synthesized from donor 11 and ac-
ceptor 18 according to the general procedure A, amorphous solid, [α]D
−20 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz, selected data): δ 1.73 (m,
2H, NHCH2CH2CH2O), 1.83 (s, 3H, CH3CO), 2.98 (br. s, 1H, OH), 3.09 (m,
2H, NHCH2CH2CH2O), 4.43 (d, 1H, J1,2 = 9.2 Hz, H-15), 4.58 (d, 1H,
J1,2 = 8.1 Hz, H-1), 4.67 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.88 (d, 1H, J1,2 = 8.4 Hz,
H-17), 4.90 (s, 1H, PhCH), 4.93 (d, 1H, J1,2 = 7.2 Hz, H-1), 4.99 (d, 1H,
J1,2 = 7.7 Hz, H-1), 5.02 (s, 1H, PhCH), 5.07 (s, 2H, PhCH2), 5.11 (s, 1H,
PhCH), 5.12 (d, 1H, J1,2 = 7.4 Hz, H-1), 5.23 (s, 1H, PhCH), 5.31 (t,
1H, J = 9.4 Hz, H-37), 5.46, 5.48, 5.61 (3 PhCH), 7.18–8.10 (m, 60H, 12 Ph).
13C NMR (CDCl3, 150 MHz, selected data): δ 20.5 (CH3CO), 29.5

8
D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
(NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 40.5, 40.6 (2 ClCH2), 66.4
(PhCH2), 98.8, 99.0, 99.2, 99.5 (2C) (5 C-1), 100.5, 101.0, 101.1 (3 PhCH),
101.3 (C-1), 101.5, 101.6 (2C), 101.7 (4 PhCH), 102.2 (C-15), 164.5, 164.7,
165.2, 165.3, 166.3, 166.7 (4 PhCO, 2 ClCH2CO), 169.4 (CH3CO). HRMS
(ESI): calcd. for C136H133Cl2NO45 [M + Na]+ 2592.7419; found 2592.7467.
101.7 (11 PhCH). HRMS (ESI): calcd. for C206H201Cl2NO69 [M + 2 NH4]2+
1949.1152; found 1949.1156.
4.18. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-2,3-di-O-
chloroacetyl-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (25)
4.16. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-2,3-di-O-
chloroacetyl-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (21)
Nonasaccharide 21 (91%) was obtained from donor 11 and ac-
ceptor 20 according to the general procedure A, amorphous solid,
[α]D −21 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz, selected data): δ 1.65
(m, 2H, NHCH2CH2CH2O), 1.79, 1.81 (2 s, each 3H, 2 CH3CO), 2.94 (br.
s, 1H, OH), 3.10 (m, 2H, NHCH2CH2CH2O), 4.41 (d, 1H, J1,2 = 8.5 Hz,
H-1), 4.57 (d, 1H, J1,2 = 8.2 Hz, H-1), 4.61 (d, 1H, J1,2 = 6.9 Hz, H-1),
4.70 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.78 (t, 1H, J1,2 = 6.7 Hz, H-2), 4.80–4.85
(m, 4H, PhCH, 2 H-1, H-2), 4.89 (t, 1H, J = 6.9 Hz, H-2), 4.92–4.97 (m,
2H, PhCH, H-1), 4.99–5.03 (m, 3H, H-1, 2 H-2), 5.03–5.07 (m, 4H,
PhCH2, 2 PhCH), 5.09–5.16 (m, 3H, H-1, H-2, PhCH), 5.22 (t, 1H,
J = 8.1 Hz, H-2), 5.24 (s, 1H, PhCH), 5.32 (t, 1H, J = 8.0 Hz, H-39), 5.45,
5.46, 6.63 (3 s, each 1H, 3 PhCH), 7.15–8.10 (m, 75H, 15 Ph). 13C NMR
(CDCl3, 150 MHz, selected data): δ 20.5, 20.6 (2 CH3CO), 29.4
(NHCH2CH2CH2O), 37.9 (NHCH2CH2CH2O), 40.5, 40.6 (2 ClCH2), 66.4
(PhCH2), 98.1, 98.4, 98.6, 98.9, 99.0, 99.4 (2C), 101.2, 102.1 (9 C-1),
100.4, 101.0, 101.1 (2C), 101.3, 101.4, 101.5, 101.6 (2C) (9 PhCH), 156.3
(OCONH), 164.5, 164.7, 165.2, 165.3, 166.3, 166.6, 166.7 (5 PhCO,
2 ClCH2CO), 168.9, 169.3 (2 CH3CO). HRMS (ESI): calcd. for
Tridecasaccharide 25 was obtained in 95% yield by coupling of
acceptor 24 with donor 11 according to the general procedure A,
amorphous powder, [α]D −17 (c 1, CHCl3). 1H NMR (CDCl3, 600 MHz,
selected data): δ 1.68 (m, 2H, NHCH2CH2CH2O), 1.80 (s, 6H, 2 CH3CO),
1.81, 1.83 (2 s, each 3H, 2 CH3CO), 2.94 (br. s, 1H, OH), 3.10 (m, 2H,
NHCH2CH2CH2O), 4.44 (d, J1,2 = 8.2 Hz, H-1), 4.59 (d, J1,2 = 7.1 Hz, H-1),
4.63 (d, J1,2 = 5.1 Hz, H-1), 4.66–4.69 (m, 2H, 2 H-1), 4.72 (d, J1,2 = 5.8 Hz,
H-1), 4.79–4.84 (m, 2H, H-1, H-2), 4.83–4.94 (m, 12H, 3 H-1, 6 H-2,
3 PhCH), 4.96, 5.10, 5.19, 5.21, 5.27, 5.47, 5.48, 5.64 (8 s, 8H, 8 PhCH),
4.99 (d, J1,2 = 6.2 Hz, H-1), 5.02–5.07 (m, 4H, H-1, 2 H-2, PhCH), 5.08
(s, 2H, PhCH2), 5.12–5.17 (m, 3H, H-1, H-2, PhCH), 5.24 (t, 1H,
J = 7.8 Hz, H-2), 5.35 (t, 1H, J = 9.3 Hz, H-313), 7.15–8.10 (m, 105H, 21
Ph). 13C NMR (CDCl3, 150 MHz, selected data): δ 20.5 (2C), 20.6 (2C),
(4 CH3CO), 29.5 (NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 40.5, 40.7
(2 ClCH2), 66.4 (PhCH2), 67.4 (NHCH2CH2CH2O), 97.9 (2C), 98.4 (3C),
98.6, 98.9, 99.1, 99.5 (3C), 101.2, 102.2 (13 C-1), 100.5, 101.0 (2C),
101.2 (3C), 101.3, 101.4 (4C), 101.6, 101.7 (2C) (13 PhCH), 156.4
(OCONH), 164.5 (2C), 164.8 (3C), 165.3 (2C), 166.2, 166.6 (7 PhCO,
2 ClCH2CO), 168.9, 169.0 (2C), 169.3 (4 CH3CO). HRMS (ESI): calcd.
for C241H235Cl2NO81 [M + 2 NH4]2+ 2272.2177; found 2272.2277.
C
171H167Cl2NO57 [M + Na]+ 3238.9469; found 3238.9473.
4.17. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-2,3-di-O-
chloroacetyl-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (23)
4.19. General procedure B. Conversion of oligosaccharides obtained
by General procedure A into new diol acceptors
Acetyl chloride (8 equiv.) was added to a solution of an oligo-
saccharide, obtained by general procedure A, and 2,4,6-collidine (15
equiv.) in CH2Cl2 (15 mL/mmol) and the mixture was stirred at rt
for 20 h. MeOH (50 equiv.) was added and stirring was continued
for next 30 min. The mixture was diluted with CH2Cl2, successive-
ly washed with aq 1 M HCl, water, satd aq NaHCO3, dried, and
concentrated. 2,4,6-Collidine (9 equiv.) and thiourea (17 equiv.) were
added to a solution of the acetylated product in EtOAc (20 mL/mmol)
and ethanol (20 mL/mmol), the resulting mixture was stirred at 80 °C
for 12 h, cooled to rt, and concentrated. A solution of the residue
in CH2Cl2 (100 mL) was washed with aq 1 M HCl, water, satd aq
NaHCO3, dried, and the solvent was evaporated. The residue was
purified by silica gel column chromatography (toluene → 85:15
toluene–acetone) to provide a diol acceptor.
Undecasaccharide 23 was synthesized by glycosylation of ac-
ceptor 22 with donor 11 according to the general procedure A in
80% yield, amorphous solid, [α]D −18 (c 1, CHCl3). 1H NMR (CDCl3,
600 MHz, selected data): δ 1.70 (m, 2H, NHCH2CH2CH2O), 1.79, 1.80,
1.83 (3 s, each 3H, 3 CH3CO), 2.93 (br. s, 1H, OH), 3.10 (m, 2H,
NHCH2CH2CH2O), 4.43 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.59 (d, 1H, J1,2 = 7.2 Hz,
H-1), 4.63 (d, 1H, J1,2 = 5.5 Hz, H-1), 4.68 (d, 1H, J1,2 = 5.2 Hz, H-1),
4.72 (d, 1H, J1,2 = 5.9 Hz, H-1), 4.79–4.93 (m, 12H, 3 H-1, 6 H-2, 2 PhCH,
NH), 4.97, 5.04, 5.10, 5.15, 5.18, 5.27, 5.47, 5.48, 5.64 (9 s, 9H, 9 PhCH),
4.99 (d, 1H, J1,2 = 6.7 Hz, H-1), 5.01–5.07 (m, 2H, H-1, H-2), 5.08 (s,
2H, PhCH2), 5.12–5.18 (m, 3H, H-1, 2 H-2), 5.23 (t, 1H, J = 7.9 Hz, H-2),
5.35 (t, 1H, J = 9.4 Hz, H-311), 7.18–8.08 (m, 90H, 18 Ph). 13C NMR
(CDCl3, 150 MHz, selected data): δ 20.4, 20.6, 20.7 (3 CH3CO), 29.6
(NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 40.5, 40.7 (2 ClCH2), 66.4
(PhCH2), 97.9, 98.1, 98.4, 98.5, 98.6, 98.8, 99.0, 99.3, 99.4, 101.2, 102.1
(11 C-1), 100.5, 100.9 (2C), 101.2 (C2), 101.3, 101.4 (2C), 101.6 (2C),
4.20. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (18)
Diol 18 was obtained from 17 according to the general proce-
dure B in 86% yield, amorphous solid, [α]D −24 (c 1, CHCl3). 1H NMR

D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
9
(CDCl3, 600 MHz, selected data): δ 1.67 (m, 2H, NHCH2CH2CH2O), 1.82
(s, 3H, CH3CO), 3.10 (m, 2H, NHCH2CH2CH2O), 4.57 (d, 1H, J1,2 = 8.0 Hz,
H-15), 4.59 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.69 (d, 1H, J1,2 = 5.4 Hz, H-1),
4.86 (t, 1H, J = 5.4 Hz, H-2), 4.88 (d, 1H, J1,2 = 6.5 Hz, H-1), 4.92 (br.
t, 1H, NH), 4.96 (s, 1H, PhCH), 4.97 (t, 1H, J = 6.8 Hz, H-2), 5.08 (s,
2H, PhCH2), 5.10 (s, 1H, PhCH), 5.23 (t, 1H, J = 8.0 Hz, H-2), 5.29 (t,
1H, J = 6.5 Hz, H-2), 5.32, 5.47, 5.64 (3 s, each 1H, 3 PhCH), 7.18–8.10
(m, 45H, 9 Ph). 13C NMR (CDCl3, 150 MHz, selected data): δ 20.5
(CH3CO), 29.4 (NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 66.4 (PhCH2),
67.3 (NHCH2CH2CH2O), 98.5 (2C), 99.5, 101.2, 102.5 (5 C-1), 101.2,
101.3, 101.4, 101.7, 101.9 (5 PhCH), 125.3–137.3 (9 Ph), 156.4
(OCONH), 164.5, 164.7, 165.5 (3 PhCO), 169.3 (CH3CO). HRMS (ESI):
calcd. for C99H99NO32 [M + Na]+ 1836.6042; found 1836.6089.
(NHCH2CH2CH2O), 97.8, 98.0, 98.2 (2C), 98.3, 98.4, 99.4 (7 C-1), 101.0
(3C, 3 PhCH), 101.3 (2C, C-1, PhCH), 101.4 (3C, 3 PhCH), 101.7, 101.8
(2 PhCH), 102.5 (C-19). HRMS (ESI): calcd. for C169H167NO56 [M + 2
NH4]2+ 1571.0464; found 1571.0489.
4.23. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (24)
4.21. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (20)
Diol 24 was obtained from 23 according to the general proce-
dure B in 86% yield, amorphous solid, [α]D −19 (c 1, CHCl3). 1H NMR
(CDCl3, 600 MHz, selected data): δ 1.67 (m, 2H, NHCH2CH2CH2O), 1.79
(s, 6H, 2 CH3CO), 1.81, 1.83 (2 s, each 3H, 2 CH3CO), 3.11 (m, 2H,
NHCH2CH2CH2O), 4.55 (d, 1H, J1,2 = 7.9 Hz, H-111), 4.59 (d, 1H,
J1,2 = 7.3 Hz, H-1), 4.63 (d, 1H, J1,2 = 5.4 Hz, H-1), 4.66–4.70 (m, 2H,
2 H-1), 4.75 (d, 1H, J1,2 = 5.1 Hz, H-1), 4.79–4.93 (m, 14H, 4 H-1, 7
H-2, 3 PhCH, NH), 4.96 (s, 1H, PhCH), 5.01 (t, 1H, J = 7.0 Hz, H-2),
5.07–5.12 (m, 4H, H-1, PhCH, PhCH2), 5.17, 5.19, 5.21 (3 s, each 1H,
3 PhCH), 5.23 (t, 1H, J = 8.0 Hz, H-2), 5.31 (t, 1H, J = 6.9 Hz, H-2), 5.37,
5.46, 5.64 (3 s, each 1H, 3 PhCH), 7.18–8.12 (m, 90H, 18 Ph). 13C NMR
(CDCl3, 150 MHz, selected data): δ 20.6 (4C, 4 CH3CO), 29.5
(NHCH2CH2CH2O), 38.0 (NHCH2CH2CH2O), 66.5 (PhCH2), 67.4
(NHCH2CH2CH2O), 97.8, 97.9, 98.1, 98.3 (3C), 98.4 (2C), 99.5 (9 C-1),
100.9, 101.0 (2C), 101.1 (4 PhCH), 101.3 (3C, C-1, 2 PhCH), 101.4 (2C),
101.5, 101.7, 101.8 (5 PhCH), 102.6 (C-111), 126.1–137.2 (18 Ph), 156.4
(OCONH), 164.5, 164.8 (3C), 164.9, 165.6 (6 PhCO), 168.9, 169.0 (2C),
169.3 (4 CH3CO). HRMS (ESI): calcd. for C204H201NO68 [M + 2 NH4]2+
1894.1489; found 1894.1480.
Diol 20 was prepared from 19 according to the general proce-
dure B in 90% yield, amorphous solid, [α]D −24 (c 1, CHCl3). 1H NMR
(CDCl3, 600 MHz, selected data): δ 1.78 (m, 2H, NHCH2CH2CH2O), 1.78,
1.81 (2 s, each 3H, 2 CH3CO), 2.72 (br. s, 1H, OH), 3.09 (m, 2H,
NHCH2CH2CH2O), 3.14 (br. s, 1H, OH), 4.55 (d, 1H, J1,2 = 8.0 Hz, H-17),
4.58 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.62 (d, 1H, J1,2 = 5.5 Hz, H-1), 4.74 (d,
1H, J1,2 = 5.4 Hz, H-1), 4.79 (t, 1H, J = 5.5 Hz, H-2), 4.82–4.86 (m, 3H,
H-1, H-2, PhCH), 4.87–4.92 (m, 3H, H-1, H-2, NH), 4.93 (s, 1H, PhCH),
4.98 (t, 1H, J = 6.9 Hz, H-2), 5.06–5.10 (m, 4H, H-1, PhCH2, PhCH),
5.23 (t, 1H, J = 8.0 Hz, H-2), 5.29 (t, 1H, J = 6.7 Hz, H-2), 5.34, 5.46,
5.64 (3 s, each 1 H, 3 PhCH), 7.18–8.08 (m, 60H, 12 Ph). 13C NMR
(CDCl3, 150 MHz, selected data): δ 20.5, 20.6 (2 CH3CO), 29.4
(NHCH2CH2CH2O), 37.9 (NHCH2CH2CH2O), 66.4 (PhCH2), 67.4
(NHCH2CH2CH2O), 98.0, 98.3, 98.4 (2C), 101.1, 102.6 (6 C-1), 99.5,
101.0, 101.7, 101.8, (4 PhCH), 101.3 (4C, C-1, 3 PhCH), 126.1–137.3
(12 Ph), 156.4 (OCONH), 154.5, 164.7, 164.8, 165.5 (4 PhCO), 169.0,
169.3 (2 CH3CO). HRMS (ESI): calcd. for C134H133NO44 [M + K]+
2498.7832; found 2498.7800.
4.24. General procedure C. Preparation of unprotected
oligoglucosides
A solution of a protected oligosaccharide (100–120 mg) in a 5:5:1
(v/v/v) mixture of CHCl3—MeOH—aq 1 M HCl (10 mL) was heated
at 40–45 °C until TLC (9:1 CHCl3—MeOH) indicated disappearance
of products with Rf > 0 (4–6 h). The mixture was made neutral with
Amberlyst A-26 (HCO3−), the resin was filtered off and thoroughly
washed with a 1:1 mixture CHCl3—MeOH (50 mL), and the com-
bined filtrates were taken to dryness. The residue was dissolved in
MeOH (10 mL), 1 M sodium methoxide (1 mL) was added, and the
mixture was heated at 40–45 °C with stirring for 4–5 h, then water
(2–5 mL) was added until homogeneity, and stirring was contin-
ued for next 6–16 h. The mixture was made neutral with Amberlite
IR-120 (H+), then Amberlyst A-26 (HCO3−) (2–3 mL) was added. The
resins were filtered off, thoroughly washed with aq 50% MeOH, and
the combined filtrates were concentrated. The residue was dis-
solved in aq MeOH (the proportion of water was increased from 30
to 70% on increasing oligosaccharide length, while water was used
for 13-mer), then Pd(OH)2/C (50–70 mg) and 1 M HCl (50–70 μL)
were added, and the resulting mixture was stirred at rt under a hy-
drogen atmosphere until TLC (10:10:3 CHCl3—MeOH—water) showed
the absence of substances with Rf > 0 (2–4 h). The catalyst was fil-
tered off through a Celite layer, washed with aq MeOH, and the
combined filtrates were taken to dryness. The residue was sub-
jected to gel-permeation chromatography, appropriate fractions were
collected and freeze-dried to provide a 3-aminopropyl β-(1→3)-
oligoglucoside as amorphous fluffy solid.
4.22. 3-Benzyloxycarbonylaminopropyl 4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (22)
Diol 22 (88%) was synthesized from 21 according to the general
procedure B, amorphous solid, [α]D −25 (c 1, CHCl3). 1H NMR (CDCl3,
600 MHz, selected data): δ 1.67 (m, 2H, NHCH2CH2CH2O), 1.81. 1.82,
1.84 (3 s, each 3H, 3 CH3CO), 2.67 (br. s, 1H, OH), 2.09 (br. s, 1H, OH),
3.10 (m, 2H, NHCH2CH2CH2O), 4.55 (d, 1H, J1,2 = 7.9 Hz, H-19), 4.59
(d, 1H, J1,2 = 7.2 Hz, H-1), 4.63 (d, 1H, J1,2 = 5.1 Hz, H-1), 4.69 (d, 1H,
J1,2 = 5.2 Hz, H-1), 4.75 (d, 1H, J1,2 = 5.0 Hz, H-1), 4.80–4.93 (m, 11H,
3 H-1, 5 H-2, NH, 2 PhCH), 4.97 (s, 1H, PhCH), 5.00 (t, 1H, J = 7.0 Hz,
H-2), 5.07–5.12 (m, 4H, H-1, PhCH, PhCH2), 5.17, 5.19 (2 s, each 1H,
2 PhCH), 5.23 (t, 1H, J = 7.9 Hz, H-2), 5.31 (t, 1H, J = 6.8 Hz, H-2), 5.37,
5.46, 5.64 (3 s, each 1H, 3 PhCH), 7.18–8.13 (m, 75H, 15 Ph). 13C NMR
(CDCl3, 150 MHz, selected data): δ 20.3, 20.5, 20.6 (3 CH3CO), 29.4
(NHCH2CH2CH2O), 38.2 (NHCH2CH2CH2O), 66.4 (PhCH2), 67.3

10
D.V. Yashunsky et al./Carbohydrate Research 419 (2016) 1–10
4.25. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (26)
1H and 13C NMR data see Tables 1 and 2. HRMS (ESI): calcd. for
C81H139NO66 [M + H]+ 2182.7624; found 2182.7606.
Trisaccharide 26 was prepared from protected precursor 13 ac-
cording to the general procedure C in 87% yield, [α]D −22 (c 1, water).
1H and 13C NMR data see Tables 1 and 2. HRMS (ESI): calcd. for
C21H39NO16 [M + H]+ 562.2342; found 562.2344.
Acknowledgment
This work was supported by a grant from the Ministry of Edu-
cation and Science of the Russian Federation (unique identification
number RFMEF157914X0022).
4.26. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (27)
References
Pentasaccharide 27 was obtained from protected pentamer 17
according to the general procedure C in 95% yield, [α]D −23 (c 1,
water). 1H and 13C NMR data see Tables 1 and 2. HRMS (ESI): calcd.
for C33H59NO26 [M + H]+ 886.3398; found 886.3395.
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4.27. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (28)
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Heptasaccharide 28 was prepared from 19 according to the
1
general procedure C in 94% yield, [α]D −16 (c 1, water). H and 13C
NMR data see Tables 1 and 2. HRMS (ESI): calcd. for C45H79NO36
[M + H]+ 1210.4455; found 1210.4438.
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4.28. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (29)
32.
11. Adamo R, Hu Q-Y, Torosantucci A, Crotti S, Brogioni G, Allan M, et al. Chem Sci
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Nonasaccharide 29 was obtained from 21 according to the general
procedure C in 81% yield, [α]D −16 (c 1, water). 1H and 13C NMR data
see Tables 1 and 2. HRMS (ESI): calcd. for C57H99NO46 [M + H]+
1534.5511; found 1534.5523.
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4.29. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (30)
25. Ananikov VP, Khokhlova EA, Egorov MP, Sakharov AM, Zlotin SG, Kucherov AV,
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Undecasaccharide 30 was prepared from precursor 23 accord-
ing to the general procedure C in 84% yield, [α]D −17 (c 1, water).
1H and 13C NMR data see Tables 1 and 2. HRMS (ESI): calcd. for
C69H119NO56 [M + H]+ 1858.6567; found 1858.6552.
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4.30. 3-Aminopropyl β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl-(1→3)-β-D-glucopyranoside (31)
38. Wang Y, Yan Q, Wu J, Zhang L-H, Ye X-S. Tetrahedron 2005;61:4313–22.
39. Gerbst AG, Grachev AA, Yashunsky DV, Tsvetkov YE, Shashkov AS, Nifantiev NE.
J Carbohydr Chem 2013;32:205–21.
Tridecasaccharide 31 was prepared from precursor 25 accord-
ing to the general procedure C in 71% yield, [α]D −16 (c 1, water).