Synthesis of an aminooxy derivative of the trisaccharide globotriose Gb3

Article abstract of DOI:10.1080/07328303.2014.925913

The synthesis of α-aminooxy trisaccharide moiety [α-d-Gal-(1,4)-β-d-Gal-(1,4)-β-d-Glc-α-aminooxy], related to the cell surface globotriaosylceramide (Gb3) receptor of the B subunit of the AB₅ Shiga toxin of Shigella dysenteriae, has been carrie

Full text of DOI:10.1080/07328303.2014.925913

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Synthesis of an Aminooxy Derivative of
the Trisaccharide Globotriose Gb3
Samir Ghosha & Peter R. Andreanaa
a Department of Chemistry and Biochemistry and School of Green
Chemistry and Engineering, University of Toledo, Toledo, OH 43606
Published online: 23 Jun 2014.
To cite this article: Samir Ghosh & Peter R. Andreana (2014) Synthesis of an Aminooxy Derivative
of the Trisaccharide Globotriose Gb3, Journal of Carbohydrate Chemistry, 33:7-8, 381-394, DOI:
10.1080/07328303.2014.925913
To link to this article: http://dx.doi.org/10.1080/07328303.2014.925913
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Journal of Carbohydrate Chemistry, 33:381–394, 2014
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2014.925913
Synthesis of an Aminooxy
Derivative of the Trisaccharide
Globotriose Gb3
Samir Ghosh and Peter R. Andreana
Department of Chemistry and Biochemistry and School of Green Chemistry
and Engineering, University of Toledo, Toledo, OH 43606
The synthesis of α-aminooxy trisaccharide moiety [α-D-Gal-(1,4)-β-D-Gal-(1,4)-β-D-Glc-
α-aminooxy], related to the cell surface globotriaosylceramide (Gb3) receptor of the B
subunit of the AB5 Shiga toxin of Shigella dysenteriae, has been carried out for the
first time in 11 steps with a 15% overall isolated yield. A highlight of this work entails
utilizing chemically compatible synthetic transformations, including those related to
glycosylation, incorporative of the succinimidyl moiety as a precursor to the aminooxy
Gb3 derivative. The fully deprotected trisaccharide aminooxy compound was reacted
with a carbonyl compound, leading to oxime formation in quantitative yield, which un-
derscores the importance for future glyco-conjugations.
Keywords Aminooxy sugar; Gb3 trisaccharide; Shiga toxin; Glycosylation
Human oligosaccharides and glycoconjugates[1–5] are well-known entities
that play critical roles in maintaining homeostatic biological processes;[6,7]
however, those that come from foreign sources (e.g., bacterial based) or that
are aberrant in nature have attracted a great deal of attention from the syn-
thetic and biological communities for a sustained period of time due to their
strong relationship in diseased states. Within the aforementioned classifica-
tions, glycosphingolipids[8–12] are known to be naturally occurring bioactive
molecules most often embedded in the membrane of all animal cells and even
in some plant cells.[13–16] Physiological functions of glycosphingolipids include
mediating intercellular interactions as well as modulating the activity of some
proteins in the plasma membrane. Accumulation of glycosphingolipids in the
Received April 2, 2014; accepted May 15, 2014.
Address correspondence to Peter R. Andreana, Department of Chemistry and Biochem-
istry and School of Green Chemistry and Engineering, University of Toledo, 2801 W.
Bancroft St, Toledo, OH 43606. E-mail: peter.andreana@utoledo.edu
381

S. Ghosh and P. R. Andreana
382
lysome can result in glycosphingolipid storage diseases such as Gaucher dis-
ease,[17,18] Fabry disease,[19,20] urinary tract infections,[21] and a host of other
complications including pediatric neurodegenerative disease.[22]
One detrimental glycolipid effect caused by Shiga toxin(s) is the loss of
millions of human lives each year. In 1897, Kiyoshi Shiga first described the
bacterial origin of dysentery caused by the bacillus Shigella dysenteriae. The
Shiga toxins have two subunits, unit A and unit B, and they belong to the AB5
toxin[23,24] family of antigens. The cytotoxic enzyme unit A, an N-glycosidase
that catalyzes rRNA depurination activity and leads to cell death, is noncova-
lently linked with a nontoxic homopentameric cell adhesion carrier B5 unit.
Each of these units has three binding sites for a specific glycolipid called globo-
triaosyl (Gb3), known as a Pk-trisaccharide.[21,25] The B5 unit mediates up-
take and intracellular transport of the toxin through specific binding to the
sugar domain of the Gb3 in the plasma membrane of cells. Gb3 also serves
as a cell surface receptor for Shiga-like toxins (SLTs).[26–30] Not surprisingly,
SLT vaccine development has been an important area of study since the early
1990s.[31] Encouragingly, there are Shiga toxin vaccine candidates currently
being pursued. One strategy utilizes live attenuated[32] Shiga toxin–producing
Escherichia coli, and another uses the carrier protein KLH conjugated to Shiga
toxin.[33] In both of the aforementioned accounts, the vaccines have been able
to produce active and functional IgG antibodies capable of neutralizing Shiga
toxin; however, a successful verdict is still pending.
In noting the importance of these carbohydrate–protein binding inter-
actions, numerous syntheses of Gb3 have been reported by prominent syn-
thetic carbohydrate groups.[34–46] Those strategies have incorporated linkers
at the anomeric position including the ceramide,[46] p-aminophenyl(pAP),[34] 6-
(p-cinnamoylphenoxy)hexyl tether,[35] and methyl glycosides.[36] In an attempt
to develop physiologically stable oxime bonds for sugar–sugar couplings for en-
tirely carbohydrate-based vaccines, our approach is to introduce the aminooxy
precursor succidimidyl group at an early stage in the synthetic endeavor, tak-
ing advantage of a flexible, functional group strategy amenable to deriviti-
zation.[47] In utilizing chemically compatible reagents, the goal is to develop
orthogonal reactions that are nontoxic, high yielding, and robust toward pre-
exposure of an aminooxy moiety for the synthesis of Gb3-α-ONH2. As far as we
are aware, Gb3-α-ONH2 has never been previously synthesized and the benign
nature and orthogonality of the reagents we have employed overcome some of
the current challenges regarding succinimidyl lability.
In this context, we describe a synthesis of a Gb3 trisaccharide deriva-
tive noted as [α-D-Gal-(1,4)-β-D-Gal-(1,4)-β-D-Glc-α-aminooxy] (1) (Fig. 1). The
synthesis of Gb3-α-ONH2 was achieved by sequential convergent glycosyla-
tions of a suitably protected monosaccharide (2) as well as disaccharide (3)
that were prepared from commercially available reducing sugars using chemi-
cally compatible reaction conditions (Fig. 2). The use of N-hydroxysuccinimide

Aminooxy Derivative of Globotriose Gb3
383
Figure 1: The Gb3-α-ONH2 (1) as an aminooxy sugar for oxime-forming conjugation.
(4) (NHS) was essential for installing the -OHN2 functionality. Other similar
types of NHS equivalents, such as N-hydroxyphthalamide (PhtOH)[48–50] and
N-pentenoyl (NHPent),[51–54] have also been used for the -ONH2 functionality,
but due to our experience with NHS, others were not utilized.[47,55]
Phenyl-β–D-galactopyranosyl-(1,4)-1-thio-β-D-glucopyranoside (3) was pr-
epared from D-lactose following a literature procedure.[46] Ethyl-2,3,4,6-tetra-
O-benzyl-1-thio-β-D-galactopyranoside (2),[56] derived from D-galactose and N-
hydroxysuccinamide (4), was commercially available. Compound 3 was treated
with benzaldehyde dimethyl acetal to protect the 4- and 6-hydroxy groups in
the presence of camphor sulphonic acid to yield compound 5, which was then
benzylated using benzyl bromide and sodium hydride, furnishing compound 6
in 81% overall isolated yield (for two steps) (Scheme 1). Stereoselective gly-
cosylation of disaccharide donor 6 with N-hydroxy succinimide 4[57] (1.5 eq.
due to low solubility in DCM) in the presence of N-iodosuccinimide (NIS) and
trimethylsilyl trifluoromethanesulfonate (TMSOTf)[58,59] yielded compound 7
in 90% as a mixture of anomers (10:1, α/β) as determined from 1H NMR. Struc-
tural confirmation of compound 7, using spectral analysis, was assigned as
follows: δ 5.56 (d, J = 4.0 Hz, 1 H, 1A), 5.51 (s, 1 H, PhCH), 4.35 (d, J = 7.9 Hz,
1 H, 1B) and 2.75 (s, 4 H, -C( O)CH2-CH2-C(O)). The 13C NMR data was as
follows: δ 101.7 (C-1A), 103.2 (C-1B), 101.7 (PhCH), 171.1 (2 C, -C(O)CH2-CH2-
C(O)), and 25.8 (2 C, -C( O)CH2-CH2-C(O)). Successful 1,2-cis-glycosylation
of the NHS derivative was supported by the coupling constant (J = 4.0 Hz) of
the anomeric H-1 in compound 7. Selective reductive ring opening of the ben-
zylidene acetal of 7 was achieved by treatment with ethereal HCl and sodium
Figure 2: Compounds used in the synthesis of target trisaccharide Gb3-α-ONH2.

S. Ghosh and P. R. Andreana
384
cyanoborohydride in THF to produce disaccharide acceptor 8 with a 4-OH ex-
posed as a single regioisomer in an overall isolated 69% yield.
Ph
O
OH
O
OH
O
OR
O
HO
HO
O
a
O
RO
b
SPh
O
SPh
O
RO
HO
RO
HO
HO
RO
3
5: R = OH
6: R = OBn
c
4
Ph
O
OBn
O
OBn
O
OBn
O
HO
BnO
O
d
O
O
N
O
N
O
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
O
O
8
7
O
O
Scheme 1: Reagents: (a) benzaldehyde dimethyl acetal, DMF, rt, overnight, 89% isolated
yield; (b) benzyl bromide, NaH, TBAB (cat.), DMF, rt, 4 h, 91% overall isolated yield; (c)
N-iodosuccinimide (NIS), trimethylsilyl trifluoromethanesulfonate, 4Å MS, CH2Cl2, –20◦C, 1 h,
81% isolated yield; (d) etheral HCl, sodium cyanborohydride, anhydrous THF, argon, 69%
isolated yield.
Scheme 2: Reagents: (a) N-iodosuccinimide, trimethylsilyl trifluoromethanesulfonic, MS 4Å,
CH2Cl2, –20◦C, 1 h, 54% isolated yield; (b) H2, 20% Pd(OH)2-C, rt, 8 h, 76% isolated yield; (c)
hydrazine monohydrate, MeOH:H2O (1:1), rt, 3 h, 68% isolated yield.
Stereoselective glycosylation of disaccharide acceptor 8 with thioglycoside
derivative 2[56] in the presence of (NIS) and TMSOTf furnished trisaccharide
1
9 in 57% yield as a mixture of anomers (20:1, α/β). Spectral H NMR data
analysis confirmed the trisaccharide formation of the 1,2-cis glycosidic linkage:
δ 5.51 (d, J = 4.0 Hz, 1 H, 1A), 5.07 (d, J = 3.5 Hz, 1 H, 1C), 4.33 (d, J =
7.74 Hz, 1 H, 1B), 2.75 (s, 4 H, -C( O)CH2-CH2-C(O)). The 13C NMR data was

Aminooxy Derivative of Globotriose Gb3
385
Scheme 3: Reagents: (a) 2-propanone, H2O, rt.
as follows: δ 171.1 (2 C, -C(O)CH2-CH2-C(O103.2 (C-1B), 101.7 (C-1A), 101.1
(C-1C), 25.9 (2 C-C( O)CH2-CH2-C(O)). Both 1,2-cis glycosidic linkages were
confirmed through the examination of coupling constants (J = 4.0 Hz) of the
H-1 of compound 8 and (J = 3.5 Hz) of the H-1 of compound 4.
In the final deprotection phase, we first utilized Pearlman’s catalyst[60]
for benzyl group removal to obtain compound 10 followed by treatment with
hydrazine hydrate[21] for deprotection of the succinimide group to furnish
the final product 1. After completion as noted by TLC, two by-products[57,61]
were also characterized, namely, acetohydrazide and tetrahydropyriddazine-
3,6-dione that are notorious to interfere with product purification. In noting
the latter to be true when using silica gel chromatography, these two by-
products were eventually removed using a Bio-gel P-2 column with water as
the eluent giving ultra-pure 1.
In order to test the reactivity of the aminooxy 1, we elected to utilize a
simple carbonyl compound in the form of a ketone. As our group has been con-
jugating to carbonyl aldehydes, such as those noted in our previous published
work,[47,55] we were eager to utilize a carbonyl ketone for three reasons: (1)
first, as a means to validate that oxime formation would occur as readily with
carbonyl ketones as our experience indicated it would with carbonyl aldehy-
des; (2) for future development of novel oxime protecting groups; and (3) for
future studies on the hydrolysis of oxime bonds. Thus, 2-propanone was used
for the oxime forming reaction that ultimately gave rise to compound 11 in
quantitative yield.
In conclusion, a convenient, stereocontrolled synthesis of Gb3-α-ONH2 was
accomplished using a succinimidyl group at the anomeric position of lactose,
which remained intact until the final step of the synthesis. The succinimidyl
group was transformed into an aminooxy moiety after global deprotection,
which readily allows for selective conjugation to carbonyl compounds. The
aminooxy Gb3 was also reacted with 2-propanone to form an oxime link, which
marks our initial attempts at developing new oxime protecting groups and for
determining stability.

S. Ghosh and P. R. Andreana
386
EXPERIMENTAL SECTION
General Experimental Methods
1H, 13C, 2D COSY, and HMQC nuclear magnetic resonance spectra were
recorded on Bruker 600 (1H NMR 600 MHz; 13C NMR 150) at ambient tem-
perature with CDCl3, D2O as solvent, and TMS as internal reference unless
1
otherwise stated. Chemical shifts are reported in δ ppm. Data for H NMR
are reported as follows: chemical shift, integration, multiplicity (s = singlet,
d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multi-
plet) and coupling constants in Hertz. All 13C NMR spectra were recorded
with complete proton decoupling. Low-resolution mass spectra (LRMS) were
acquired on a Waters Acuity Premiere XE TOF LC-MS using electrospray
ionization.
All reactions were carried out in oven-dried glassware under an ar-
gon atmosphere unless otherwise noted. Reactions were monitored by thin
layer chromatography over silica gel-coated TLC plates. TLC was visual-
ized by warming ceric sulphate (2% Ce(SO4)2 in 2 N H2SO4)-sprayed plates
on a hot plate. Column chromatography was performed on silica gel 60
(0.040–0.063 mm). All chemicals were of commercial grade and were used with-
out further purification. Anhydrous solvents were obtained from Aldrich and
EMD. Yields refer to chromatographically and spectroscopically pure material
unless otherwise noted.
Phenyl-(2,3-di-O-benzyl-4,6-O-benzylidene-β–D-galactopyranos-
yl)-(1→4)-2,3,6-tri-O-benzyl-1 thio- β-D-glucopyranoside (6)
To a solution of compound 3 (2 g, 4.60 mmol) in anhydrous DMF (20 mL)
were added benzaldehyde dimethyl acetal (0.84 g, 5.52 mmol) and camphor
sulfonic acid, and the reaction mixture was allowed to stir overnight at rt. The
reaction was quenched with triethylamine and solvents were removed under
reduced pressure. The crude material 5 was dissolved in CH2Cl2 (50 mL) and
the organic layer was washed three times with water, dried using Na2SO4,
concentrated, and dried under vacuum. To a solution of the dried product in
anhydrous DMF (30 mL) were added powdered NaH (at 60% w/w dispersion
in mineral oil) (1.8 g, 45 mmol), benzyl bromide (3.8 mL, 32 mmol), and tetra-
butylammonium bromide (200 mg, 0.62 mmol) and the reaction mixture was
allowed to stir briskly at rt for 4 h. The reaction was quenched by adding satd.
NH4Cl and concentrated under reduced pressure. The crude mass was dis-
solved in CH2Cl2 (150 mL) and the organic layer was washed with water, dried
using Na2SO4, and finally concentrated. The crude product was purified over
SiO2 using hexane-EtOAc (7:1) as eluant to give pure compound 6 (3.28 g,
81%).

Aminooxy Derivative of Globotriose Gb3
387
1H NMR (600 MHz, CDCl3): δ 7.61–7.21 (m, 35 H, Ar-H), 5.50 (s, 1 H,
PhCH), 5.29 (d J = 10.4 Hz, 1 H, PhCH2), 4.89–4.76 (m, 7 H, PhCH2), 4.69
(d, J = 9.8 Hz, 1 H, 1A), 4.60–4.58 (d, J = 11.9 Hz, 1 H, PhCH2), 4.53 (d, J =
7.9 Hz, 1 H, 1B), 4.40 (1 H, PhCH2), 4.30–4.26 (m, 1 H, 6B), 4.09–4.08 (d, J =
3.7 Hz, 1 H, 4B), 4.06 (t, J1/2, 2/3 = 9.6 Hz, 1 H, 4A), 3.97–3.95 (dd, J1/2,1,3 = 3.9,
11.0 Hz, 1 H, 6A), 3.91–3.89 (m, 1 H, 6B), 3.84–3.79 (m, 2 H, 6A, 2B), 3.70 (t, J
= 8. 9 Hz, 1 H, 3A), 3.52 (t, J = 9.9 Hz, 1 H, 2A), 3.48–3.46 (dd, J1/2,1,3 = 3.7,
9.6 Hz, 1 H, 3B), 3.4–3.40 (m, 1 H, 5A), 3.04 (m, 1 H, 5B).
13C NMR (150 MHz, CDCl3): δ 139.0–126.8 (Ar-C), 103.1 (1B), 101.6
(PhCH), 87.6 (1A), 85.3 (3A), 80.3 (2A), 79.9 (3B), 79.6 (5A), 79.1 (2B), 77.4 (4A),
76.3 (OBn), 75.6 (OBn), 73.9 (4B), 73.2 (OBn), 71.9 (OBn), 69.2 (6B), 68.6 (6A),
66.7 (5B). ESI-MS (LR MS) [C60H60O10SNa]+ found 995.9.
Succinamidyl-(2,3-di-O-benzyl-4,6-O-benzylidene-β–D-galacto-
pyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (7)
To a solution of compound 6 (3.28 g, 3.71 mmol) and compound 4 (N-
hydroxysuccinamide) (1.28 g, 11.12 mmol) in anhydrous CH2Cl2 (50 mL) was
˚
added MS-4A (2.00 g,) and the reaction mixture was allowed to stir at rt for 1 h
under argon. The reaction mixture was cooled to –20◦C and N-iodosuccinimide
(NIS; 1.00 g, 4.45 mmol) and TMSOTf (80 μL) were then added. After stirring
at the same temperature for 3 h, the reaction mixture was quenched with sat-
urated NaHCO3 and sodium thiosulfate, filtered through a pad of celite, and fi-
nally washed with CH2Cl2 (100 mL). The organic layer was then washed three
times with aqueous Na2SO4 and water in succession, dried using Na2SO4, and
concentrated under reduced pressure to give the crude product, which was
purified over SiO2 using hexane-EtOAc (5:1) as the eluant to furnish pure α
anomeric product 7 (2.96 g, 81%).
1H NMR (600 MHz, CDCl3): δ 7.58–7.16 (m, 30 H, Ar-H), 5.56 (d, J =
4.0 Hz, 1 H, 1A), 5.51 (s, 1 H, PhCH), 5.23 (d, J = 10.4 Hz, 1 H, PhCH2), 5.02
(d, J = 11.3 Hz, 1 H, PhCH2), 5.03–4.77 (m, 6 H, PhCH2), 4.76–4.75 (m, 1 H,
5A), 4.52 (d, J = 11.9 Hz, 1 H, PhCH2), 4.35 (d, J = 7.9 Hz, 1 H, 1B), 4.30–4.27
(dd, J1/2,1,3 = 1.2, 12.4 Hz, 1 H, 6B), 4.25 (d, J = 11.9 Hz, 1 H, PhCH2), 4.13–4.11
(dd, J1/2,1,3 = 2.3, 11.2 Hz, 1 H, 6A), 4.08–4.05 (m, 3 H, 3A, 4A, 4B), 3.92–3.90
(dd, J1/2,1,3 = 1.7, 12.4 Hz, 1 H, 6B), 3.83–3.80 (dd, J1/2,1,3 = 7.9, 9.6 Hz, 1 H,
2B), 3.76–3.73 (m, 1 H, 2A), 3.52–3.50 (dd, J1/2,1,3 = 1.9, 11.2 Hz, 1 H, 6A),
3.34–3.32 (dd, J1/2,1,3 = 7.9, 9.6 Hz, 1 H, 3B), 2.96 (s, 1 H, 5B), 2.75 (s, 4 H, -C(
O)CH2-CH2-C(O)).
13C NMR (150 MHz, CDCl3): δ 171.1 (2 C, -C(O)CH2-CH2-C(O)),
139.3–126.9 (Ar-C), 103.2 (1B), 101.7(PhCH), 101.7 (1A), 79.8 (3A), 78.7 (3B),
78.1 (2A), 77.0 (4A), 76.4 (OBn), 75.9 (OBn), 74.0 (4B), 73.4 (OBn), 73.2 (OBn),
72.6 (5A), 72.0 (OBn), 69.3 (6B), 68.0 (6A), 66.6 (5B), 25.8 (2 C, -C( O)CH2-CH2-
C(O)). ESI-MS (LR MS) [C58H59NO13Na]+ found 1000.2.

S. Ghosh and P. R. Andreana
388
Succinamidyl-(2,3,6-tri-O- benzyl-α–D-galactopyranosyl)-(1→4)-
2,3,6-tri-O-benzyl-β-D-glucopyranoside (8)
To a solution of compound 7 (2.96 g, 3.02 mmol) in THF (10 mL), sodium
cyanoborohydride (0.95 g, 15.13 mmol) was added and stirred for 1 h under an
atmosphere of argon, and then the reaction mixture was cooled to ice temper-
ature and treated with ethereal HCl until gas evolution ceased. The reaction
mixture stirred for 15 min in the same temperature and then slowly warmed
to rt and allowed to stir for 1 h further. After completion, the reaction mixture
was filtered through a pad of celite. The filtrate was concentrated and purified
on a silica gel column using hexane/EtOAc (2:1) as an eluent to give pure 8
(2.04 g, 69%).
1H NMR (600 MHz, CDCl3): δ 7.53–7.23 (Ar-H), 5.56 (d, J = 4.0 Hz, 1 H,
1A), 5.06–5.01 (m, 2 H, PhCH2), 4.88–4.76 (m, 6 H, PhCH2), 4.75–4.74 (m, 1
H, 5A), 4.57–4.55 (m, 2 H, PhCH2), 4.47–4.45 (d, J = 12.0 Hz, 1 H, PhCH2),
4.35–4.34 (d, J = 7.8 Hz, 1 H, 1B), 4.34–4.33 (d, J = 11.9 Hz, 1 H, PhCH2),
4.09–3.98 (m, 4 H, 4A, 4B, 6A1, 3A), 3.76–3.74 (dd, J1/2,1,3 = 7.0, 9.8 Hz, 1 H,
6B1), 4.71–3.69 (dd, J1/2,1,3 = 4.0, 9.8 Hz, 1 H, 2A), 3.66–3.63 (dd, J1/2,1,3 = 8.0,
9.1 Hz, 1 H, 2B), 3.60–3.57 (m, 1 H, 6B2), 3.56–3.54 (m, 1 H, 6A2), 3.36–3.33 (m,
2 H, 3B), 2.73 (s, 4 H, -C( O)CH2-CH2-C(O)).
13C NMR (150 MHz, CDCl3): δ 171.0 (2 C, -C(O)CH2-CH2-C(O)),
139.2–127.4 (Ar-C), 102.6 (1B), 101.4 (1A), 81.1 (3B), 79.5 (3A), 79.1 (2B), 77.8
(2A), 75.7 (OBn), 75.6 (OBn), 75.5 (4A), 73.6 (OBn), 73.2 (OBn), 73 1 (OBn), 73.0
(5B), 72.4 (5A), 72.1 (OBn), 68.6 (6B), 67.5 (6A), 66.3 (4B), 25.4 (2 C-C(O)CH2-
CH2-C(O)). ESI-MS (LR MS) [C58H61NO13Na]+ found 1002.9.
Succinamidyl-(2,3,4,6-tetra-O-benzyl-α–D-galactopyranosyl)-
(1→4)-(2,3,6-tri-O- benzyl-β–D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (9)
To a solution of compound 8 (2.04 g, 2.08 mmol) and compound 2 (1.58 g,
˚
2.70 mmol) in anhydrous CH2Cl2 (40 mL) was added MS-4A (1.00 g) and the
reaction mixture was allowed to stir at rt for 1 h under argon. The vessel was
then cooled to –10◦C, and N-iodosuccinimide (NIS; 0.73 g, 3.24 mmol) and TM-
SOTf (40 μL) were added. After stirring at a constant temperature for 4 h,
the reaction mixture was quenched with saturated NaHCO3 and sodium thio-
sulfate, filtered through a pad of celite, and washed three times with CH2Cl2
(100 mL). The organic layer was washed with aq. Na2SO4 and water in suc-
cession, dried using Na2SO4, and then concentrated under reduced pressure
to give the crude product. Purification ensued over SiO2 using hexane-EtOAc
(2:1) as the eluent to furnish pure 9 (1.69 g, 54%).
1H NMR (600 MHz, CDCl3): δ 7.49–7.14 (m, 50 H, Ar-H), 5.51 (d, J =
4.0 Hz, 1 H, 1A), 5.13 (J = 10.9 Hz, 1 H, PhCH2), 5.07 (d, J = 3.5 Hz, 1
H, 1C), 4.94 (J = 11.4 Hz, 1 H, PhCH2), 4.89 (J = 11.1 Hz, 1 H, PhCH2),

Aminooxy Derivative of Globotriose Gb3
389
4.85–4.70 (m, 7 H, PhCH2), 4.70–4.69 (d, J = 9.9 Hz, 1 H, 5A), 4.56–4.45 (m,
5 H, PhCH2), 4.40–4.37 (m, 1 H, 5C), 4.33 (d, J = 7.74 Hz, 1 H, 1B), 4.34–4.31
(m, 2 H, PhCH2), 4.27–4.25 (d, J = 11.9 Hz, 1 H, PhCH2), 4.21–4.19 (dd, J1/2,1,3
= 8.4, 9.6 Hz, 1 H, 6B1), 4.15–4.10 (m, 3 H, 6B1, 2 PhCH2), 4.07 (brs, 1 H, 2C),
4.05–4.00 (m, 4 H, 4A, 4B, 4C, 3A), 3.99–3.96 (t, J = 9.3 Hz, 1 H, 3A), 3.67–3.64
(m, 2 H, 2A, 2B), 3.57–3.55 (dd, J1/2,1,3 = 5.3, 9.6 Hz, 1 H, 6B2), 3.53–3.50 (t, J
= 9.0 Hz, 1 H, 6C1), 3.49–3.47 (m, 1 H, 6A), 3.30–3.28 (m, 1 H, 5B), 3.23–3.21
(dd, J1/2,1,3 = 4.7, 8.6 Hz, 1 H, 3B), 3.20–3.18 (q, 1 H, 6C), 2.75 (s, 4 H, -C(
O)CH2-CH2-C(O)).
13C NMR (150 MHz, CDCl3): δ 171.1 (2 C, -C(O)CH2-CH2-C(O)),
139.5–127.5 (Ar-C), 103.2 (1B), 101.7 (1A), 101.1 (1C), 81.9 (3B), 79.7 (3A), 79.7
(3C), 79.3 (2B), 77.9 (2A), 76.9 (2C), 76.5 (4C), 76.0 (OBn), 75.9 (OBn), 75.5 (4A),
75.2 (OBn), 75.1 (4B), 74.0 (OBn), 73.7 (5B), 73.5 (OBn), 73.4 (OBn) , 73.3 (OBn),
72.7(OBn), 72.6 (5A), 72.4, (OBn), 69.7 (5C), 68.2 (6B), 68.1 (6C), 67.8 (6A),
25.9 (2 C-C( O)CH2-CH2-C(O)). ESI-MS (LR MS) [C92H95NO18Na]+ found
1525.4.
Succinamidyl-(α–D-galactopyranosyl)-(1→4)-(β–D-
galactopyranosyl)-(1→4)-β-D-glucopyranoside (10)
To a solution of compound 9 (1.69 g, 1.12 mmol) in CH3OH (30 mL) was
added 20% Pd(OH)2-C (200 mg) and the mixture was allowed to stir at rt un-
der a positive pressure of hydrogen for 10 h. The reaction mixture was filtered
through a pad of celite and then washed with CH3OH-H2O (60 mL; 1:3 v/v).
The combined filtrate was evaporated under reduced pressure to furnish com-
pound 10, which was purified through a Sephadex LH-20 column using H2O
as an eluent to give pure compound 10 (514 mg, 76%).
1H NMR (600 MHz, CDCl3): δ 5.63 (d, J = 4.0 Hz, 1 H, 1A), 5.15 (J =
3.6 Hz, 1 H, 1C), 4.72 (J = 7.7 Hz, 1 H, 1B), 4.62 (d, J = 10.2 Hz, 1 H, 5A),
4.53 (m, 1 H, 5B), 423–4.21 (m, 2 H, A4, B4), 4.15–4.09 (m, 4 H, C3, C5, A6,
C6), 4.05–3.95 (m, 4 H, A2, A3, C2, B6, C6), 3.94–3.89 (m, 4 H, B3, C4, A6, B6),
3.80–3.78 (m, 1 H, B2), 3.00 (s, 4 H, -C( O)CH2-CH2-C(O)).
13C NMR (150 MHz, CDCl3): δ 175.1 (2 C, -C( O)CH2-CH2-C(O)),103.4
(1B), 103.3 (1A), 100.5 (1C), 77.3 (B4), 75.4 (C4), 78.3 (C5), 72.7 (A5), 72.4
(B3), 71.2 (B2), 71.1 (2 C, A4, B5), 70.6 (A3), 69.4 (A2), 69.2 (C2), 68.8 (C3),
25.6 (2 C-C( = O)CH2-CH2-C(O)). ESI-MS (LR MS) [C22H35NO18Na]+ found
623.9.
Aminooxy-(α–D-galactopyranosyl)-(1→4)-(β–D-
galactopyranosyl)-(1→4)-β-D-glucopyranoside (1)
To a solution of compound 10 (514 mg, 0.85 mmol) in H2O (20 mL) was
added hydrazine hydrate and the mixture was allowed to stir at rt for 4 h. After
completion of the reaction (TLC solvent (Rf 0.3) [4:3:1, n-butanol:H2O:AcOH]),

S. Ghosh and P. R. Andreana
390
H2O was evaporated under reduced pressure to furnish compound 1, which
was purified through a P-2 Biogel column using H2O as the eluent to give pure
compound 1 (302 mg, 68%).
1H NMR (600 MHz, CDCl3): δ 4.98 (d, J = 4.0 Hz, 1 H, 1A), 4.92 (J =
3.9 Hz, 1 H, 1C), 4.49 (J = 7.8 Hz, 1 H, 1B), 4.34–4.32 (m, 2 H, 4A, 4B), 3.92–3.87
(m, 4 H, C5, A6, C6, A5), 3.82–3.79 (m, 3 H, C2, A6, B6), 3.77–3.74 (m, 1 H, C4),
3.73–3.71 (m, 1 H, A3, B3), 3.67–3.64 (m, 3 H, B6, C6, C5), 3.62–3.60 (m, 1 H,
A2), 3.57–3.54 (m, 1 H, B2).
13C NMR (150 MHz, CDCl3): δ 103.1 (1B), 101.2 (1A), 100.2 (1C), 78.3 (C5),
77.3 (B4), 75.4 (C4), 72.1 (A3), 71.5 (B3), 70.8 (B2), 70.7 (A2), 70.5 (C2), 70.4 (A4),
68.9 (B5), 68.5 (C3). ESI-MS (LR MS) [C18H33NO16Na]+ found 542.0.
ACKNOWLEDGMENT
S.G. and P.R.A. would like to acknowledge Dr. Yong Wah Kim (University of
Toledo) for NMR assistance.
FUNDING
This work was supported by the National Institute of Health (NCI-R01
CA156661-01).
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