D-Glucose and d-mannose-based metabolic probes. Part 3: Synthesis of specifically deuterated d-glucose, d-mannose, and 2-deoxy-d-glucose

Article abstract of DOI:10.1016/j.carres.2012.11.021

Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80 years ago. Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect, clearly differentiates malignant from non-maligna

Full text of DOI:10.1016/j.carres.2012.11.021

Carbohydrate Research 368 (2013) 111–119
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
D-Glucose and D-mannose-based metabolic probes. Part 3: Synthesis
of specifically deuterated D-glucose, D-mannose, and 2-deoxy-D-glucose
Izabela Fokt a, Stanislaw Skora a, Charles Conrad b, Timothy Madden a, Mark Emmett c,
Waldemar Priebe a,
⇑
a Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Box 1950, Houston, TX 77030, USA
b Department of Neuro-Oncology, 1515 Holcombe Blvd., Houston, TX 77030, USA
c Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch Galveston, 301 University Boulevard, Galveston, TX 77555, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2012
Received in revised form 22 November 2012
Accepted 23 November 2012
Available online 13 December 2012
Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80 years ago.
Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect,
clearly differentiates malignant from non-malignant cells. The resurgence of interest in cancer metabo-
lism aims at a better understanding of the metabolic differences between malignant and non-malignant
cells and the creation of novel therapeutic and diagnostic agents exploiting these differences.
Modified D-glucose and D-mannose analogs were shown to interfere with the metabolism of their
respective monosaccharide parent molecules and are potentially clinically useful anticancer and diagnos-
tic agents.
Keywords:
2-Deutero-
2-Deutero-
6-Deutero-
6-Deutero-
2-Deutero-2-deoxy-
Metabolic probes
D
D
D
D
-glucose
-mannose
-glucose
-mannose
One such agent, 2-deoxy-D-glucose (2-DG), has been extensively studied in vitro and in vivo and also
clinically evaluated. Studies clearly indicate that 2-DG has a pleiotropic mechanism of action. In addition
to effectively inhibiting glycolysis, 2-DG has also been shown to affect protein glycosylation. In order to
better understand its molecular mechanism of action, we have designed and synthesized deuterated
D-glucose
molecular probes to study 2-DG interference with
spectrometry. We present here the synthesis of all desired probes: 2-deutero-
mannose, 6-deutero- -glucose, 6-deutero- -mannose, and 2-deutero-2-deoxy- -glucose as well as their
complete chemical characterization.
D-glucose and
D-mannose metabolism using mass
D
-glucose, 2-deutero-D-
D
D
D
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
under normoxic conditions. Diagnostic and therapeutic opportuni-
ties stem from the fact that, in contrast to malignant cells, normal
cells under normoxia will preferentially use aerobic glucose
metabolism to produce energy (via the Krebs/TCA cycle), while gly-
colysis, the anaerobic metabolism of D-glucose, will be used only
when cells are deprived of oxygen (hypoxic conditions). This pro-
vides a potentially wide therapeutic window.
The replacement of the hydroxyl at C-2 with the fluorine atom
in 2-FDG does not interfere with the first step of glycolysis and al-
lows for hexokinase-mediated phosphorylation at the C-6 hydroxyl
Metabolism of simple monosaccharides plays an important role
in tumor growth and survival. Unlike normal cells, which reserve
the anaerobic metabolism of sugars for hypoxic conditions, tumor
cells preferentially metabolize monosaccharides via the ancient
anaerobic pathway of glycolysis regardless of ambient oxygen
tension.
Given this metabolic discrepancy, modified D-glucose and D-
mannose analogs have been developed to exploit the metabolism
of malignant cells. These modified analogs are known to interfere
with the metabolism of their respective monosaccharides, and
are thus potentially clinically useful anticancer and diagnostic
to 2-FG 6-phosphate; however, further metabolism to D-fructose 6-
phosphate is not possible. Increased accumulation of 18F-labeled 2-
FDG 6-phosphate inside the highly glycolytic tumor cells can be
easily detected with the use of PET.
agents. For example, 18F-labeled 2-deoxy-2-fluoro-
D-glucose
(2-18FDG) is now widely used in oncology as a diagnostic and
imaging agent in conjunction with positron emission tomography
(PET).1
The therapeutic option of blocking glycolysis, the primary
means of energy-generating glucose metabolism in tumor cells,
has been clinically explored using 2-deoxy-
D-arabino-hexose (2-
2-FDG works by exploiting the eponymous Warburg effect,2
that is the increased dependence of tumor cells on glycolysis even
deoxy-
D
-glucose, 2-DG)3 and has led to numerous in vitro studies.4
Like 2-FDG, the lack of a hydroxyl group at C-2 in 2-DG leads to the
accumulation of 2-DG 6-phosphate inside highly glycolytic tumor
⇑
cells and the blockade of
that, for historical reasons, 2-DG has been called 2-deoxy-
D
-glucose metabolism. It should be noted
Corresponding author. Tel.: +1 713 792 3777; fax: +1 713 745 4975.
E-mail addresses: wpriebe@mdanderson.org, wp@wt.net (W. Priebe).
D-glu-
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.11.021

112
I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
cose, even though it is also a 2-deoxy-
sharing the same structural feature, the 2-DG can also affect D-
mannose metabolism, including glycosylation processes. 2-DG
has been demonstrated to affect glycosylation processes5 and in-
duce endoplasmic reticulum stress.6
D
-mannose and, because of
hydroxyl groups in positions 1, 3, 5, and 6 because that allowed
us to achieve one-step deprotection of the final product. The key
step in our process was to obtain benzyl -glucoside with ketone
group in position 2. Reduction of the ketone with NaBD4 led exclu-
sively to the desired gluco product (8).
a
In order, to study in detail the molecular aspects of the effects of
Specifically, benzyl 3-O-benzyl-4,6-benzylidene-a-D-mannopy-
2-DG on the metabolism of D-glucose and D-mannose using mass
ranoside (5) used as the starting compound was obtained using
spectrometry, we have designed specifically deuterated probes
and performed their synthesis. We present in detail chemical syn-
methods described in the literature. Briefly D-mannose was
transformed into its benzyl glycoside,10 followed by formation of
4,6-benzylidene acetal,11 and benzylation at position 3 utilizing a
stannyl ester.11 The free 2-hydroxyl group was oxidized with high
yield (64%) using Dess–Martin periodinate. Reduction of such
obtained ketone (6) with NaBD4 gave benzyl 3-O-benzyl-4,6-ben-
thesis and characterization of 2-deutero-
-mannose (13), 6-deutero- -glucose (19), 6-deutero-
-glucose (4).
D-glucose (8), 2-deutero-
D
D
D-mannose
(26), and 2-deutero-2-deoxy-
D
zylidene-2-deutero-a-D-glucopyranose (7). Hydrogenation of (7)
2. Results and discussion
with hydrogen and palladium on carbon Degussa type allowed us
to obtain high purity product in gram scale. Our approach required
fewer synthetic steps than the method described by Haines. The
final deprotection step was fast, with easy work-up and purification
of the final compound, and may be performed in multigram scale.
2.1. Synthesis of 2-deutero-2-deoxy-
D-glucose
We proposed an efficient method for synthesis of 2-deutero-2-
deoxy-
deoxy-
D
D
-glucose. Current efficient methods led to 2-deutero-2-
-mannose with an axially oriented deuterium atom rather
2.3. Synthesis of 2-deutero-D-mannose
than to 2-deutero-2-deoxy-
tero-2-deoxy-
-mannose by Wong and Gray7 utilized appropri-
ately protected ketene dithioacetal from -glucose and its
reduction with lithium aluminum deuteride, and final deprotection
to 2-deutero-2-deoxy- -mannose. Yet another method described
by Lehmann and Petry8 started from fructose that, in a five-step
process, led to 2-azi-2-deoxy- -arabino-hexitol that, when irradi-
ated at 350 nm in water gave 2-deutero-2-deoxy- -mannose. Both
D-glucose. The first synthesis of 2-deu-
D
Our approach (Scheme 3) toward synthesis of 2-deutero-
D-
D
mannose (13) was based on the reported stereoselective reduction
of b-D-arabino-hexopyranoside-2-ulose derivatives with NaBH4
D
compounds with manno configuration.12 Our goal was to prepare
b-glycoside with a ketone group at C-2 position, with all remaining
hydroxyl groups protected with benzyl ether or benzylidene ace-
tals. We applied the Lichtenthaler synthetic process leading in
D
D
methods are multistep processes and are not useful for preparing a
multigram amount of final product.
Looking for the method that would be easiest to perform on a
multigram scale, we decided to use commercially available and
two-steps from 2-acetoxy-D-glucal with good yield to respective
b-ketone.13
The starting compound 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-D-
glucose (9), was prepared from per-O-benzylated-D-glucal by its
inexpensive per-O-acetylated
The whole process is presented in Scheme 1. The per-O-acetylated
-glucal was deacetylated in standard Zemplén condition, and
D-glucal as the starting material.
cis-hydroxylation14 followed by acetylation. Compound (9) was
reacted with HBr (33% solution in acetic acid) to give glycosyl
bromide which upon treatment with DBU formed 2-O-acetoxy-
D
without purification was per-O-benzylated. The addition of D2O
in the presence of DBr in tetrahydrofuran gave 3,4,6-tri-O-ben-
3,4,6-tri-O-benzyl-
described by Lichtenthaler,12 2-acetoxy-
formed into a ketone (11). Reduction of (11) using NaBD4 gave
benzyl 3,4,6-tri-O-benzyl-b- -mannopyranoside (12) with high
yield (90%). Hydrogenation of (12) with hydrogen and palladium
on carbon Degussa type gave the final compound, 2-deutero-
D-glucal (10). Subsequently, using the method
D
-glucal (10) was trans-
zyl-2-deutero-D-glucose (3).
Proton nuclear magnetic resonance (1H NMR) analysis demon-
strated that the obtained product had only equatorial deuterium
atom. Debenzylation of (3) by hydrogenation using a Paar appara-
tus led to the final product with a total yield of 42%. The described
method may be easily scaled up, if needed.
D
D-
mannose (13). Each step in the described method can be scaled
up to a multigram scale.
2.2. Synthesis of 2-deutero-D-glucose
2.4. Synthesis of 6-deutero-D-glucose
The most commonly described methods in the literature for
preparation of 2-deutero- -glucose (8) are the enzymatic process
D
Synthesis of (S)-
D
-6-deutero-glucose was already described by
-(6,6-
Xu15 and used as a substrate for methyl 2,3,4-tri-O-benzyl-
D
with milligram amounts of final products. The only synthetic route
for preparation of (8) was described by Haines.9 Starting from
2H2)glucopyranoside which upon Swern oxidation of 6-OH group
and subsequent reduction of an aldehyde with nondeuterated
(R)-(+)-Alpine-Borane gave with high optical purity an (S) isomer.
As there was no need in our studies for pure (S) or (R) stereoiso-
mers; we opted for a rather simpler high yield synthesis of
6-deutero-glucose (19) as an (S/R) mixture, as shown in
Scheme 4.
methyl 3-O-benzoyl-4,6-benzylidene-a-D-glucopyranoside in a
five-step process, Haines obtained milligram amounts of (8), which
was per-O-acetylated and analyzed as its per-acetylated derivative.
No analytical data for free (8) was included. In our approach
(Scheme 2), we chose glucose as a starting material. We decided
to use benzyl ethers and benzylidene acetals for protection of
O
O
O
OH
D
O
OH
D
AcO
AcO
BnO
BnO
BnO
BnO
HO
d)
a)
b)
c)
HO
OBn
OH
(4)
OAc
OBn
(1)
Scheme 1. Synthesis of 2-deoxy-2-deutero-
(2)
(3)
D
-glucose. Reagents and conditions: (a) 1 N MeONa/MeOH; (b) NaH, BnBr, DMF; (c) DBr/D2O, THF; (d) Pd/C, H2, THF, EtOH.

I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
113
O
OBn
OH
O
OBn
O
O
OBn
OH
O
O
O
a)
b)
Ph
Ph
Ph
O
O
O
D
OBn
(5)
OBn
(6)
OBn
(7)
O
OH
HO
c)
OH
D
HO
OH
(8)
Scheme 2. Synthesis of 2-deoxy-2-deutero-D-glucose. Reagents and conditions: (a) Dess–Martin periodinate, CH2Cl2; (b) NaBD4, CH2Cl2, MeOH; (c) Pd/C, H2.
O
OAc
OAc
O
BnO
BnO
BnO
BnO
c)
d)
a)
b)
OAc
OBn
OBn
(9)
(10)
O
OBn
O
O
OBn
O
OH
D
BnO
BnO
BnO
HO
e)
f)
D
HO
BnO
OH
OH
OBn
(11)
OBn
(12)
OH
(13)
Scheme 3. Synthesis of 2-deutero-D-mannose. Reagents and conditions: (a) HBr/AcOH, CH2Cl2; (b) DBU, CH2Cl2; (c) NBS, EtOH, CH2Cl2; (d) BnOH, Ag2CO3, CH2Cl2; (e) NaBD4,
CH2Cl2, MeOH; (f) Pd/C, H2.
To economize synthesis of 19 a mixture of
a
and b anomers 14
hydrogenation gave 6-deutero-D-glucose (19) as a mixture of (6R)
was used as a starting material. Benzyl ,b- -glucopyranoside (14)
a
D
and (6S) epimers.
protected with 6-O-tert-butyldimethylsilyl ether was benzylated
to give compound 15 and then desilylated to benzyl 2,3,4-tri-O-
2.5. Synthesis of 6-deutero-D-mannose
benzyl-
in the literature thus for analytical purposes
were separated and characterized as 16 and 16b. Swern oxidation
of the primary hydroxyl group at C-6 gave the aldehyde 17 that
without separation was subjected to reduction with sodium boro-
deuteride to give compound 18 deuterated at position C-6. The
same oxidation and reduction reactions were performed using
D
-glucopyranoside (16). Product 16 has not been described
a
and b anomers 16
We found no evidence in the literature of a method for the syn-
thesis of 6-deutero- -mannose. Since we did not seek to obtain
pure (R) or (S) isomer, we decided to use a strategy similar to that
a
D
used for preparation of 6-deutero-
shown in Scheme 5.
D-glucose. Our approach is
Starting from benzyl
a-D-mannopyranoside (20) protection of
pure anomers 16
a
and 16b in order to secure analytical data for
the 6-hydroxyl group with tert-butyldimethylsilyl was followed
the new chemical entities. Subsequent debenzylation of 18 by
by benzylation of the remaining hydroxyl groups and deprotection
O
OBn
OH
O
OBn
OBn
O
OBn
OBn
Me2t-BuSiO
Me2t-BuSiO
BnO
HO
BnO
a)
b)
HO
HO
OBn
OBn
(14)
(15)
(16)
D
D
O
OBn
OBn
O
OBn
O
OH
O
HO
BnO
HO
c)
d)
e)
BnO
OBn
HO
OH
OBn
(17)
OBn
(18)
HO
(19)
Scheme 4. Synthesis of 6-deutero-D-glucose. Reagents and conditions: (a) NaH, BnBr, DMF; (b) H2SO4, MeOH; (c) Swern oxidation; (d) NaBD4, CH2Cl2, MeOH; (e) Pd/C, H2.

114
I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
O
OBn
OBn
O
OBn
OH
O
OBn
OH
Me2t-BuSiO
BnO
Me2t-BuSiO
HO
a)
b)
HO
HO
OBn
OH
OH
(20)
(21)
(22)
D
O
OBn
OBn
O
OBn
HO
BnO
O
OBn
OBn
O
d)
e)
c)
HO
BnO
BnO
OBn
OBn
OBn
(24)
OBn
(25)
(23)
D
O
OH
OH
HO
f)
HO
OH
(26)
Scheme 5. Synthesis of 6-deutero-D-mannose. Reagents and conditions: (a) t-BuMe2SiCl, imidazole, DMF; (b) NaH, BnBr, DMF; (c) H2SO4, MeOH; (d) Swern oxidation; (e)
NaBD4, CH2Cl2, MeOH; (f) Pd/C, H2.
of the position 6 to give benzyl 2,3,4-tri-O-benzyl-
a
-
D
-mannopy-
uncorrected. Optical rotations were measured using Perkin Elmer
341 Polarimeter (Perkin Elmer). Thin–layer chromatography
(TLC) was performed on an aluminum sheet coated with SilicaGel
60 F254 (EMD Millipore). Compounds were visualized on TLC plates
by spraying the plates with 20% H2SO4 in EtOH and gently heating
them. Silica gel column chromatography was performed with a
CombiFlash Companion purification system (ISCO, Inc.) and a Bio-
tage SP1 purification system (Biotage, LLC).
ranoside (23). Swern oxidation of a primary hydroxyl group at C-
6 gave an aldehyde (24) with a high yield as indicated by TLC.
Reduction of (24) with sodium borodeuteride gave benzyl 2,3,4-
tri-O-benzyl-6-deutero-
a-D-mannopyranoside as a mixture of (R)
and (S) isomers (25). Subsequent debenzylation of (25) by hydro-
genation gave the desired 6-deutero-D-mannose (26).
3. Conclusions
4.2. Benzylation—General procedure (A)
To study 2-DG interference with
metabolism, five deuterated molecular probes, namely 2-deute-
D-glucose and D-mannose
4.2.1. 3,4,6-Tri-O-benzylo-
D-glucal (2)
D-Glucal (0.184 mol) was dissolved in DMF (300 mL). The ob-
ro-
D
-glucose (8), 2-deutero-
D-mannose (13), 6-deutero-
D
D
-glucose
-glucose
tained solution was cooled down to À20 °C and NaH (60% suspen-
sion in mineral oil) (0.83 mol, 33.2 g) was added. After 5 min,
benzyl bromide (0.83 mol, 97.5 mL) was added dropwise with vig-
orous stirring. The reaction mixture was stirred while the temper-
ature was allowed to rise to ambient. Progress of the reaction was
monitored by the TLC method. After the reaction was completed,
acetic acid (0.278 mol, 16 mL) in water (100 mL) was added drop-
wise. The mixture was then diluted with water (500 mL) and the
product was extracted with hexanes (2 Â 300 mL).
(19), 6-deutero-
D
-mannose (26), and 2-deutero-2-deoxy-
(4) were synthesized in gram scale using modified or novel meth-
odology. The described synthetic procedures are practical and
scalable.
4. Experimental
4.1. Materials and methods
Unless otherwise noted, reagents were purchased from com-
mercial sources and used without further purification. DBr/D2O
solution was purchased from Sigma–Aldrich and has isotopic pur-
ity of 99%+, NaBD4 was purchased from Cambridge Isotope Labora-
tories (D4 99%, chemical purity 90–95%). 1H NMR spectra were
recorded on Bruker Avanti 300 or Bruker Avanti 500-MHz spec-
trometers in CDCl3 with TMS or D2O as the internal reference stan-
dard. 13C NMR spectra were recorded on a Bruker Avanti 500-MHz
spectrometer in CDCl3 or DMSO-d6 with TMS as the internal refer-
ence standard or D2O. 1H and 13C signals for fully deprotected com-
pounds were assigned based on 2D spectra: proton–proton
Combined organic solutions were washed with water
(3 Â 200 mL), and dried over anhydrous Na2SO4. The solids were
filtered off and solvent was evaporated to dryness. The remaining
crude product was purified by low-pressure column chromatogra-
phy using hexanes–ethyl acetate gradient. Fractions containing
product were pooled together and evaporated to dryness, and
residual solvents were removed by additional drying with a high-
vacuum oil pump, resulting in 70 g of white crystalline product
(2) (yield 92%). Analytical data are consistent with the literature.16
4.2.2. 3,4,6-Tri-O-benzylo-2-deoxy-2-deutero-D-glucopyranose
correlation
(GRADCOSY)
and
proton–carbon
correlation
(3)
(C13HSQC) and are shown in Tables 1 and 2, respectively. Mass
spectrometry analysis was performed using Acuity UPLC-MS/MS
(TQD-3) and AMD-604 mass spectrometers. Melting points were
measured on a Büchi melting point apparatus (Büchi) and were
3,4,6-Tri-O-Benzylo-D-glucal (2) (70 g, 0.168 mol) was dissolved
in THF (400 mL). A solution of DBr in D2O (48 wt %) (0.182 mol,
21 mL) was added and the reaction mixture was stirred at room
temperature until all substrate disappeared (as determined by

I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
115
Table 1
1H NMR data for
a
and b anomers of deutero-sugarsa
Compound
H-1
H-2
H-3
H-4
H-5
H-6
H-6
J1,2
J2,3
J3,4
J4,5
J5,6 ; J5,6
J6,6
J6,6
4
a
5.29
3.4 Hz
4.95
9.8 Hz
5.25
1.70
11.9 Hz
1.51
10.9 Hz
—
3.95
3.38
9.0 Hz
3.29
9.4 Hz
3.39–3.46 m
3.89–3.77
m
3.43–3.36 m
3.89–3.77
m
3.91
12.4 Hz
3.81–3.74 m
3.89–3.77 m
3.77–3.70 m
3.95–3.84 m
3.95–3.84 m
3.95–3.86 m
3.95–3.86 m
––
9.2 Hz
3.77–3.70
m
3.73
8.9 Hz
3.50
8.8 Hz
3.86
9.7 Hz
3.67
9.6 Hz
3.73
9.6 Hz
3.51
4b
8a
3.82–3.89 m
3.51–3.46 m
8b
4.66
5.20
4.90
—
—
—
3.39–3.46 m
3.81–3.74 m
3.80–3.72 m
3.80–3.72 m
13
a
3.68
9.5 Hz
3.59
9.7 Hz
3.45–3.39 m
3.84
9.2 Hz
3.40
9.2 Hz
3.85
5.2 Hz
3.48
5.7 Hz
3.86
5.8 Hz
3.42
13b
19
19b
26
26b
a
5.25
3.8 Hz
4.66
8.0 Hz
5.22
3.55
9.2 Hz
3.26
9.2 Hz
3.99–4.00 m
3.83
—
3.76
—
3.79
—
3.75
—
3.45–3.39 m
––
9.1 Hz
3.99
9.5 Hz
3.70
a
3.69
9.7 Hz
3.61
—
4.97
3.99–4.00 m
—
9.5 Hz
9.8 Hz
6.2 Hz
a
All spectra recorded in D2O.
Table 2
(m, 2H, H-6), 3.50 (dd, 1H, J = 9.5 Hz, J = 9.2 Hz, H-4
1H, OH
H-2 ), 1.55 (dd, 1H, J = 11.4 Hz, J = 9.8 Hz, H-2b).
a
), 2.76 (br s,
13C NMR spectra of deutero-sugarsa
a
), 2.27 (d, 1H, J = 5.0 Hz, OHb), 1.67 (br d, 1H, J = 11.3 Hz,
a
Compound
C-1
C-2
C-3
C-4
C-5
C-6
JC,D
JC,D
4.2.3. Synthesis of 2-deoxy-2-deutero-
General procedure B
D
-glucopyranose (4)—
4
a
93.36
91.26
95.85
92.04
95.50
95.13
92.04
95.86
95.50
95.14
36.94
19.8 Hz
39.17
19.7 Hz
71.05
22.0 Hz
73.68
22.0 Hz
71.79
21.7 Hz
72.31
21.7 Hz
71.43
67.91
70.42
72.70
75.68
71.66
74.47
72.73
75.71
71.72
74.52
71.17
70.84
69.64
69.59
68.35
68.11
69.61
69.56
68.34
68.10
72.00
75.99
71.41
75.90
73.89
77.64
71.32
75.83
74.52
77.55
60.73
4b
60.98
60.75
60.60
63.49
62.49
3,4,6-Tri-O-benzylo-2-deoxy-2-deutero-
D-glucopyranose (3)
(11.8 g, 27 mmol) was dissolved in the mixture of THF and metha-
nol (1:2, v/v) (120 mL); Pd/C (10% Pd, Degussa type) (0.8 g) was
added and the mixture was hydrogenated using a Paar apparatus
for 25 min at room temperature under hydrogen pressure of
42 psi. After 25 min, the mixture was filtered through CeliteÒ, sol-
vent was evaporated to dryness and subsequently purified by col-
umn chromatography using a chloroform–methanol gradient for
elution. Fractions containing product were pooled together and
evaporated to dryness. The residual solvent was removed using a
high-vacuum oil pump, resulting in 3.72 g of white crystals of
8a
8b
13
a
13b
19
19b
26
26b
a
60.42
20.2 Hz
60.25
20.2 Hz
62.16
23.0 Hz
62.16
74.09
72.16
72.69
product (4) (yield 83%); mp 117–119 °C, [
a]
+47.0 (c = 1.88
D
a
H2O). HRMS calcd for C6H11DO5 [M+Na]+ m/z 188.06; found, m/z
188.05.
23.0 Hz
a
4.2.4. Synthesis of benzyl 3-O-benzyl-4,6-benzylidene
All spectra recorded in D2O.
a-
D
-arabino-hexos-2-ulo-pyranoside (6)
Dess–Martin periodinane (70 g, 165 mmol) was added to the
solution of benzyl 3-O-benzyl-4,6-benzylidene- -mannopyrano-
TLC). The reaction was quenched by the addition of solid Na2CO3
(91 mmol, 9.65 g) followed by water (50 mL). The mixture was stir-
red for an additional 15 min, and then THF was evaporated to dry-
ness. AcOEt (500 mL) was added to the remaining oily product.
Layers were separated, and the organic layer was washed with
water (150 mL), then brine (150 mL), and then dried over anhy-
drous Na2SO4. The drying agent and solvents were removed to give
a crude product that was purified by column chromatography
using hexanes–ethyl acetate gradient. Fractions containing product
were pooled together and evaporated to dryness, and then crystal-
lized from the hexanes–ethyl acetate mixture, resulting in 40.2 g of
white crystals of product (3) (yield 56%) as an inseparable anomer-
a-D
side (5) (25.9 g, 57.8 mmol) in dry CH2Cl2 (300 mL) and the ob-
tained mixture was stirred at room temperature for 48 h (TLC
control). The reaction mixture was diluted with CH2Cl2 (200 mL)
and was washed with a saturated solution of K2CO3 (3 Â 200 mL)
and then with water, until neutral pH. The solution was dried over
anhydrous Na2SO4. Inorganic salts and solvents were removed and
the product was purified by column chromatography using hex-
anes–ethyl acetate gradient for elution. Fractions containing prod-
uct were pooled together and evaporated to dryness. The residual
solvent was removed using a high-vacuum oil pump to give 16.5 g
of pure product (6) (yield 64%). Mp 131–133 °C lit.17 115–117 °C;
ic mixture. Mp 100–101 °C. [
a
]
+48.1 (c = 1.06, CDCl3). HRMS
[a] [a] +42 (c = 1.15, CDCl3). HRMS
+42.4 (c = 1.14, CDCl3). Lit.17
D D
D
calcd for C27H29DO5 [M+Na]+ m/z 458.21; found, m/z 458.42.
calcd for C27H26O6 [M+Na]+ m/z 469.16; found, m/z 469.36.
1H NMR (300 MHz, CDCl3): d 7.53 –7.24 (m, 15H, H-arom), 5.56
(s, 1H, CH benzylidene), 4.93 (d, 1H, J = 13.8 Hz, CH2), 4.77 (d, 1H,
J = 11.7 Hz, CH2), 4.71 (d, 1H, J = 12.0 Hz, CH2), 4.63 (d, 1H,
J = 11.7 Hz, CH2), 4.58 (d, 1H, J = 10.3 Hz, H-2), 4.25–4.18 (m, 2H,
1H NMR (300 MHz, CDCl3): d 7.37–7.14 (m, 15 H, H-arom), 5.39
(br s, 1H, H-1
J = 10.9 Hz, CH2b), 4.75 (dd, 1H, J = 9.5 Hz, J = 5.9 Hz, H-1b), 4.70–
4.47 (m, 5H, CH2 ,b), 4.09–3.98 (m, 2H, H-3 , H-5 ), 3.75–3.60
a), 4.89 (d, 1H, J = 10.9 Hz, CH2a), 4.88 (d, 1H,
a
a
a

116
I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
H-5, H-6), 3.89 (dd, 1H, J = 10.2 Hz, J = 9.3 Hz, H-4), 3.80 (dd, 1H,
J = 9.9 Hz, J = 9.8 Hz, H-6).
J = 10.7 Hz, H-6), 3.73 (dd, 1H, J = 10.7 Hz, J = 3.5 Hz, H-6), 2.07 (s,
3H, OAc).
4.2.5. Synthesis of benzyl 3-O-benzyl-4,6-benzylidene
4.2.8. Synthesis of benzyl 3,4,6-tri-O-benzyl-b-
D-arabino-hexos-
a-
D
-glucopyranoside (7)
2-ulo-pyranoside (11)
Benzyl 3-O-benzyl-4,6-benzylidene
a-D
-arabino-hexos-2-ulo-
A mixture of 2-acetoxy-3,4,6-tri-O-benzyl-D-glucal (10) (6.6 g,
pyranoside (6) (24.1 g, 54 mmol) was dissolved in a mixture of
CH2Cl2 (80 mL) and methanol (100 mL). Sodium borodeuteride
(2.3 g, 55 mmol) was added to the solution in 4 portions. The reac-
tion mixture was stirred at room temperature for 20 min. Solvents
were partially removed until the product started to precipitate. The
resulting white crystals were filtered and washed with methanol
and water, then dried to give 22.9 g of (7) (yield 94%); mp 114–
13.9 mmol), molecular sieves 3 Å, in CH2Cl2 (66 mL), and ethanol
(20 mmol, 1.2 mL) was prepared, cooled down to 0 °C, and stirred
for 10 min. NBS (3 g, 16.7 mmol) was added and stirring was con-
tinued until yellow color appeared. The solids were filtered off, and
the filtrate was washed with ice-cold 10% solution of Na2S2O3 and
then with water, and the remaining organic solution was dried
over anhydrous Na2SO4. The drying agent was filtered off and the
solvent was evaporated to dryness. The residue was dissolved in
CH2Cl2 (25 mL) and added to a previously prepared mixture of ben-
zyl alcohol (2.8 mL, 27.8 mmol), CH2Cl2 (50 mL), Ag2CO3 (11.5 g,
41.7 mmol), and molecular sieves 4 Å (1 g). The obtained mixture
was stirred at room temperature for 1 h, and then filtered through
CeliteÒ. The volume of the mixture was reduced to ꢀ30 mL by
evaporation. Upon addition of hexanes (200 mL) the product (11)
was precipitated, filtered, washed with hexanes and dried to give
115 °C; [
a]
+83.3 (c = 1.09, CDCl3). HRMS calcd for C27H27DO6
D
[M+Na]+ m/z 472.18; found, m/z 472.53.
1H NMR (300 MHz, CDCl3): d 7.53 – 7.23 (m, 15H, H-arom), 5.57
(s, 1H, CH benzylidene), 5.03 (s, 1H, H-1), 4.96 (d, 1H, J = 11.5 Hz,
CH2), 4.81 (d, 1H, J = 11.5 Hz, CH2), 4.77 (d, 1H, J = 11.7 Hz, CH2),
4.59 (d, 1H, J = 11.7 Hz, CH2), 4.23 (dd, 1H, J = 10.0 Hz, J = 4.6 Hz,
H-6), 3.88 (ddd, 1H, J = 10.1 Hz, J = 9.9 Hz, J = 4.6 Hz, H-5), 3.86 (d,
1H, J = 9.5 Hz, H-3), 3.75 (dd, 1H, J = 10.1 Hz, J = 10.2 Hz, H-6),
3.66 (dd, 1H, J = 9.4 Hz, J = 9.3 Hz, H-4), 2.27 (s, 1H, OH).
4.2 g of pure 11 (yield 56%). Mp 129–130 °C; [
a
]
À70.2 (c = 1.01,
D
CDCl3). HRMS calcd for C34H34O6 [M+Na]+ m/z 561.23; found, m/z
561.52.
4.2.6. Synthesis of 2-deutero-
Pd(OH)2/C (20% Pd(OH)2, Degussa type) (1.1 g) was added to the
solution of benzyl 3-O-benzyl-4,6-benzylidene- -glucopyrano-
D-glucose (8)
1H NMR (300 MHz, CDCl3): d 7.40–7.16 (m, 20H, H-arom), 4.97
(d, 1H, J = 11.4 Hz, CH2), 4.93 (d, 1H, J = 12.0 Hz, CH2), 4.75 (s, 1H,
H-1), 4.73 (d, 1H, J = 12.0 Hz, CH2), 4.62–4.52 (m, 4H, CH2), 4.19
(d, 1H, J = 8.9 Hz, H-3), 3.90 (dd, 1H, J = J = 9.0 Hz, H-4), 3.81 (ddd,
1H, J = 9.0 Hz, J = 4.9 Hz, J = 2.3 Hz, H-5), 3.76 (dd, 1H, J = 10.7 Hz,
J = 2.3 Hz, H-6), 3.72 (dd, 1H, J = 10.7 Hz, J = 5.1 Hz, H-6).
a-D
side (7) (17 g, 37.8 mmol) in THF (80 mL). The mixture was hydro-
genated using a Paar apparatus for 1 h (42 psi); methanol (100 mL)
was then added and the mixture was hydrogenated for an addi-
tional 12 h. The reaction mixture was filtered through Celite, the
solvents were evaporated, and crude product was purified by col-
umn chromatography using a chloroform–methanol gradient for
elution. Fractions containing product were pooled together and
evaporated to dryness. The residual solvent was removed using a
high-vacuum oil pump, resulting in 5.3 g of (8) as white crystals
4.2.9. Synthesis of benzyl 3,4,6-tri-O-benzyl-b-D-
mannopyranoside (12)—General procedure C
Benzyl 3,4,6-tri-O-benzyl b-D-arabino-hexos-2-ulo-pyranoside
(11) (3.5 g, 6.5 mmol) was dissolved in CH2Cl2 (15 mL), followed
by addition of methanol (15 mL), then NaBD4 (1.1 g, 27 mmol)
was added and the reaction mixture was stirred at room tempera-
ture, while progress of the reaction was monitored by TLC. After
the reaction was completed, the reaction mixture was diluted with
CH2Cl2 (75 mL), and washed with water until neutral. The crude
product was purified by column chromatography using hexanes–
ethyl acetate gradient for elution. Fractions containing product
were combined and evaporated to dryness. The residual solvents
were removed using a high-vacuum oil pump to give 3.2 g of
(yield 77%); mp 144–148 °C, [
a] +50 (c = 1.57, H2O); HRMS calcd
D
for C6H11DO6 [M+Na]+ m/z 204.06; found, m/z 204.05.
4.2.7. Synthesis of 2-acetoxy-3,4,6-tri-O-benzyl-
D-glucal (10)
The 1,2-di-O-acetyl-3,4,6-tri-O-benzyl- -glucopyranose (9)
D
(14.0 g, 26.2 mmol) was dissolved in CH2Cl2 (140 mL) then a solu-
tion of HBr in acetic acid (30 wt %) (9.5 mL, 52.3 mmol) was added.
The reaction mixture was stirred at room temperature until all
substrate was converted into glycosyl bromide (as determined by
TLC). The reaction mixture was diluted with CH2Cl2 (140 mL) and
washed with an ice-cold saturated solution of sodium carbonate
then with water until neutral, and with brine. It was then filtered
through a layer of anhydrous Na2SO4. The mixture’s volume was
reduced to about 140 mL and DBU (6 mL, 52.3 mmol) was added;
the resulting mixture was stirred at room temperature while pro-
gress of the reaction was monitored by TLC. After the reaction was
completed the reaction mixture was diluted with CH2Cl2 (100 mL)
and washed with water. The organic layer was dried over anhy-
drous Na2SO4. The drying agent and solvent were removed and
the product was purified by column chromatography using hex-
anes–ethyl acetate gradient for elution. The fractions containing
products were combined and evaporated to dryness. The residual
solvents were removed using high-vacuum oil pump to give 6.8 g
of (10) as a colorless oil which solidify upon standing (yield 55%).
(12) as a colorless oil (yield 90%); [
a
]
À53.4 (c = 1.17, CDCl3).
D
1H NMR (300 MHz, CDCl3): d 20H, H-arom), 4.95 (d, 1H,
J = 12.0 Hz, CH2), 4.89 (d, 1H, J = 10.9 Hz, CH2), 4.75 (d, 1H,
J = 10.9 Hz, CH2), 4.70–4.52 (m, 5H, CH2), 4.45 (s, 1H, H-1), 3.88
(d, 1H, J = 9.3 Hz, J = 9.4 Hz, H-4), 3.81 (dd, 1H, J = 10.8 Hz,
J = 2.2 Hz, H-6), 3.74 (dd, 1H, J = 10.8 Hz, J = 5.2 Hz, H-6), 3.54 (d,
1H, J = 9.0 Hz, H-3), 3.42 (ddd, 1H, J = 9.5 Hz, J = 5.2 Hz, J = 2.2 Hz,
H-5), 2.45 (s, 1H, OH)
4.2.10. Synthesis of 2 b-
D
-mannopyranose (13)
-mannopyranoside (12) (3 g,
Benzyl 3,4,6-tri-O-benzyl-b-
D
5.53 mmol) was dissolved in a mixture of THF–methanol (1:1 v/
v) (30 mL). Pd/C (10% Pd, Degussa type) (230 mg) was added and
the mixture was hydrogenated using a Paar apparatus (42 psi H2
pressure) for 4 h. The mixture was filtered through CeliteÒ, the fil-
trate was evaporated to dryness and the crude product was puri-
fied by column chromatography using chloroform–methanol
gradient for elution. Fractions containing product were pooled to-
gether and evaporated to dryness. The residual solvents were re-
moved using high-vacuum oil pump to give 0.942 g of white
[a] [a] + 24° (c = 1.6, CDCl3); HRMS
+21.9° (c = 1.38, CDCl3) lit.18
D D
calcd for C29H30O6 [M+Na]+ m/z 497.19; found, m/z 497.44.
1H NMR (300 MHz, CDCl3): d 7.38 –7.23 (m,15H, H-arom), 6.49
(br s, 1H, H-1), 4.57 (d, 1H, J = 11.5 Hz, CH2), 4.63 (d, 1H, J = 11.5 Hz,
CH2), 4.59 (d, 1H, J = 11.5 Hz, CH2), 4.56 (s, 2H, CH2), 4.51 (d, 1H,
J = 11.5 Hz, CH2), 4.44 (d, 1H, J = 5.1 Hz, H-3), 4.26–4.19 (m, 1H,
H-5), 3.97 (dd, 1H, J = 7.2 Hz, J = 5.1 Hz, H-4), 3.83 (dd, 1H,
crystals of product (13) (yield 94%). Mp 125–129 °C, [a] +13.5°
D

I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
117
(c = 1.75, H2O); HRMS calcd for C6H11DO6 [M+Na]+ m/z 204.06;
found, m/z 204.05.
H-2), 3.36 (ddd, 1H, J = 9.6 Hz, J = 4.6 Hz, J = 2.8 Hz, H-5), 1.84 (dd,
1H, J = 7.5 Hz, J = 6.1 Hz, OH).
Benzyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside (16a): Mp 86–
4.2.11. Synthesis of benzyl 2,3,4-tri-O-benzyl-6-tert-butyldime-
87 °C [
a
]
D
+70.1° (c = 1.80, CHCl3); HRMS calcd for C34H36O6
thylsilyl-D-glucopyranoside (15)
[M+Na]+ m/z 563.24; found, m/z 563.54.
A solution of benzyl 6-tert-butyldimethylsilyl-
D
-glucopyranoside
1H NMR (300 MHz, CDCl3): d 7.42–7.24 (m, 20H, H-arom), 5.01
(d, 1H, J = 10.9 Hz, CH2), 4.89 (d, 1H, J = 11.0 Hz, CH2), 4.84 (d, 1H,
J = 10.9 Hz, CH2), 4.80 (d, 1H, J = 3.7 Hz, H-1), 4.67 (dd, 3H,
J = 12.2 Hz, J = 12.7 Hz, CH2), 4.56 (d, 1H, J = 11.9 Hz, CH2), 4.55
(d, 1H, J = 12.3 Hz, CH2), 4.07 (dd, 1H, J = J = 9.3 Hz, H-3), 3.75–
3.60 (m, 3H, H-6, H-6, H-5), 3.54 (dd, 1H, J = 9.1 Hz, H-4), 3.50
(dd, 1H, J = 9.5 Hz, J = 3.7 Hz, H-2), 1.57 (br s, 1H, OH).
(14) (8.2 g, 21.5 mmol) in DMF (150 mL) was prepared and cooled to
À15 °C. NaH (60% suspension in mineral oil) (3.1 g, 77.7 mmol) was
added and the mixture was stirred for 5 min, then benzyl bromide
(9.2 mL, 77.7 mmol) was added dropwise. The mixture was stirred
vigorously while temperature was allowed to rise to ambient. After
the reaction was completed (TLC) the reaction mixture was cooled
down to 0 °C; then acetic acid (0.755 mL, 13, 2 mmol) followed by
water (200 mL) was added. The mixture was extracted with hexanes
(250 mL). The hexane solution was washed with water
(2 Â 100 mL), then dried over anhydrous Na2SO4. Drying agent and
solvent were removed and product was purified by column chroma-
tography using hexanes–ethyl acetate gradient for elution. Fractions
containing product were pooled together and evaporated to dryness,
residual solvents were removed using a high-vacuum oil pump to
give 13 g of (15) (yield 92%). HRMS calcd for C40H50O6Si [M+Na]+
m/z 677.33; found, m/z 677.90.
4.2.13. Synthesis of benzyl 2,3,4-tri-O-benzyl-6-deutero-D-
glucopyranoside (18)
Benzyl
2,3,4-tri-O-benzyl-D-glucopyranoside
(16)
(5 g,
9,24 mmol) was oxidized using method reported in the literature19
and, without purification such obtained crude aldehyde (17) was
subjected to the reduction with NaBD4 (general procedure C). After
the reaction was completed, the reaction mixture was diluted with
CH2Cl2 (100 mL) and washed with water until neutral. The crude
product was purified by column chromatography using hexanes–
ethyl acetate gradient for elution. Fractions containing product
were combined and evaporated to dryness. The residual solvents
were removed using a high-vacuum oil pump to give 4.94 g of
(18) (yield 90%).
1H NMR (500 MHz, CDCl3): d 7.42–7.24 (m, 20H, H-arom), 5.0–
4.75 (m, H CH2
CH2- ,b), 4.49 (d, 1H, J = 7.7 Hz, H-1b), 4.05 (dd, 1H, J = 9.3 Hz,
H-3 ), 3.89 (d, 1H, J = 10.6 Hz, H-6b), 3.84 (dd, 1H, J = 11.4 Hz,
J = 4.2 Hz, H-6b), 3.77 (dd, 1H, J = 11.3 Hz, J = 3.4 Hz, H-6 ), 3.62
(br d, 1H, J = 12.2 Hz, H-6 ), 3.71–3.67 (m, 1H, H-5 ), 3.67–3.57
(m, 2H, H-3b H-4b), 3.54 (dd, J = 9.7 Hz, J = 9.4 Hz, H-4 ), 3.50
(dd, 1H, J = 9.7 Hz, J = 3.9 Hz, H-2 ), 3.47 (dd, 1H, J = 9.6 Hz,
a,b), 4.81 (d, 1H, J = 3.8 Hz, H-1a), 4.75–4.52 (m, H,
a
a
a
Benzyl 2,3,4-tri-O-benzyl-6-deutero-a-D-glucopyranoside
a
a
(18a): Mp 86–87 °C, [a] +70.2° (c = 1.27, CHCl3); HRMS calcd for
D
a
C
34H35DO6 [M+Na]+ m/z 564.25; found, m/z 564.55.
a
1H NMR (500 MHz, CDCl3): d 7.42–7.24 (m, 20H, H-arom), 5.01
J = 8.2 Hz, H-2b), 3.29 (m, H-5b), 0.92, 0.89 (2s, 9H ea, t-Bu),
(d, 1H, J = 10.8 Hz, CH2), 4.88 (d, 1H, J = 11.0 Hz, CH2), 4.84 (d, 1H,
J = 10.8 Hz, CH2), 4.81 (d, 1H, J = 3.5 Hz, H-1), 4.68 (d, 2H,
J = 11.0 Hz, CH2), 4.64 (d, 1H, J = 11.0 Hz, CH2), 4.56 (d, 1H,
J = 11.0 Hz, CH2), 4.55 (d, 1H, J = 10.8 Hz, CH2), 4.07 (dd, 1H,
J = J = 9.3 Hz, H-3), 3.75–3.62 (m, 2H, H-6, H-5), 3.34 (dd, 1H,
J = J = 9.3 Hz, H-4), 3.59 (dd, 1H, J = 9.7 Hz, J = 3.6 Hz, H-2), 1.54
(dd, 1H, J = 3.5 Hz, OH).
0.102, 0.089, 0.051, 0.04 (4s, 3H ea, Me)
13C NMR (500 MHz, CDCl3): d 139.06, 138.82, 138.76, 138.69,
138.61, 138.45, 137.69, 137.49 (C-arom), 128.55, 128.53, 128.51,
128.49, 128.40, 128.34, 128.31, 128.24, 128.20, 128.13, 128.04,
128.01, 127.92, 127.88, 127.84, 127.75, 127.73, 127.72, 127.66,
(C-arom), 102.47 (C-1b), 95.31 (C-1
82.34 (C-3 ), 80.51 (C-2 ), 77.99 (C-4
5b), 75.96, 75.93, 75.21, 75.14, 75.04, 73.10, 71.18, 70.97, 68.86
(CH2Ph), 71.98 (C-5 ), 62.46, 62.39 (C-6 b), 26.22, 26.12 (t-BuSi),
-4.79, À4.93, À5.12, À5.16 (Me2Si).
a), 84.88 (C-3b), 82.74 (C-2b),
a
a
a), 77.82 (C-4b), 75.99 (C-
13C NMR (500 MHz, CDCl3): d 138.95, 138.28, 138.25, 137.22,
(C-arom), 128.56, 128.51, 128.58, 128.47, 128.44, 128.14, 128.00,
127.95, 127.87, 127.66 (C-arom), 95.71 (C-1), 82.04 (C-3), 80.19
(C-2), 77.58 (C-4), 75.77 (CH2), 75.15 (CH2), 73.13 (CH2), 77.11
(C-5), 69.34 (CH2), 61.52 (t, J = 21 Hz, C-6).
a
a
4.2.12. Synthesis of benzyl 2,3,4-tri-O-benzyl-D-glucopyranoside
Benzyl 2,3,4-tri-O-benzyl-6-deutero-b-D-glucopyranoside
(16)
(18b): Mp 101–103 °C, [
a
]
D
À8.6° (c = 1.42, CHCl3); HRMS calcd
Sulfuric acid (1 mL, 18.8 mmol) was added to the vigorously
for C34H35DO6 [M+Na]+ m/z 564.25; found, m/z 564.55.
stirred suspension of benzyl 2,3,4-tri-O-benzyl-6-tert-butyldi-
methylsilyl-D-glucopyranoside (15) (10 g, 15.3 mmol) in methanol
1H NMR (500 MHz, CDCl3): d 7.40–7.24 (m, 20H, H-arom), 4.94
(d, 1H, J = 10.6 Hz, CH2), 4.92 (d, 1H, J = 10.2 Hz, CH2), 4.91 (d, 1H,
J = 11.8 Hz, CH2), 4.86 (d, 1H, J = 11.0 Hz, CH2), 4.80 (d, 1H,
J = 11.0 Hz, CH2), 4.72 (d, 1H, J = 11.0 Hz, CH2), 4.69 (d, 1H,
J = 11.8 Hz, CH2), 4.63 (d, 1H, J = 11.0 Hz, CH2), 4.56 (d, 1H,
J = 7.8 Hz, H-1), 3.85 (dd, 1H, J = 5.5 Hz, J = 2.5 Hz, H-6 (R or S)),
3.68 (dd, 1H, J = 9.2 Hz, J = 9.0 Hz, H-3), 3.70–3.66 (m, 1H, H-6 (R
or S)), 3.57 (dd, 1H, J = 9.5 Hz, J = 9.0 Hz, H-4 (R or S)), 3.56 (dd,
1H, J = 9.43 Hz, J = 9.2 Hz, H-4 (R or S)), 3.49 (dd, 1H, J = 8.8 Hz,
J = 8.1 Hz, H-2), 3.36 (dd, 1H, J = 9.6 Hz, J = 4.6 Hz, H-5), 1.81 (d,
1H, J = 7.7 Hz, OH).
(100 mL). The reaction mixture was stirred at room temperature.
After the reaction was completed (as determined by TLC), the reac-
tion mixture was diluted with water (100 mL). A stoichiometric
amount of Na2CO3 was added, and the product was extracted with
ethyl acetate (250 mL). The extract was washed with water and
dried over Na2SO4. The product was purified by column chroma-
tography using hexanes–ethyl acetate gradient. Fractions contain-
ing product were pooled together and evaporated to dryness,
residual solvents were removed using a high-vacuum oil pump
to give 7 g of product (16) (yield 85%).
13C NMR (500 MHz, CDCl3): d 138.66, 138.46, 138.13, 137.41,
(C-arom), 128.57, 128.47, 128.44, 128.21, 128.13, 128.03, 128.01,
127.98, 127.95, 127.76, 127.71 (C-arom), 102.94 (C-1), 84.66 (C-
3), 82.45 (C-2), 77.70 (C-4), 75.76 (CH2), 75.19 (C-5), 75.12 (CH2),
75.05 (CH2), 71.70 (CH2), 61.80 (t, J = 20.5 Hz, C-6).
Benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranoside (16b): mp 101–
103 °C [
a
]
À8.5° (c = 1.53, CHCl3), HRMS calcd for C34H36O6
D
[M+Na]+ m/z 563.24; found, m/z 563.54.
1H NMR (300 MHz, CDCl3): d 20H, H-arom), 4.99–4.60 (m, 8H,
CH2), 4.57 (d, 1H, J = 7.8 Hz, H-1), 3.87 (ddd, 1H, J = 11.9 Hz,
J = 5.9 Hz, J = 2.8 Hz, H-6), 3.70 (ddd, 1H, J = 11.9 Hz, J = 7.5 Hz,
J = 4.7 Hz, H-6), 3.67 (dd, 1H, J = 9.0 Hz, J = 8.7 Hz, H-3), 3.57 (dd,
1H, J = 9.4 Hz, J = 9.0 Hz, H-4), 3.49 (dd, 1H, J = 8.8 Hz, J = 7.8 Hz,
4.2.14. Synthesis of 6-deutero-
Benzyl 2,3,4-tri-O-benzyl-6-deutero-
(16.5 g, 30 mmol) was debenzylated according to general proce-
D-glucopyranoside (19)
D
-glucopyranoside (18)

118
I. Fokt et al. / Carbohydrate Research 368 (2013) 111–119
dure B. After purification, 4.11 g of (19) was obtained (yield 76%)
mp 148–151 °C, [ +52.2° (c = 1.53, H2O).
moved using a high-vacuum oil pump to give 3.1 g of product
(23) as a colorless oil (yield 93%). [ +53.4° (c = 1.2, CHCl3).
Lit.20
+54° (c = 0.3, CHCl3).
a]
a]
D
D
[a]
D
4.2.15. Synthesis of benzyl 6-tert-butyldimethylsilyl-
a-
D-
1H NMR (500 MHz, CDCl3): d 7.37–7.24 (m, 20H, H-arom), 4.94
(d, 1H, J = 10.8 Hz, CH2), 4.91 (d, 1H, J = 1.1 Hz, H-1), 4.75 (d, 1H,
J = 12.3 Hz, CH2), 4.69–4.60 (m, 5H, CH2), 4.43 (d, 1H, J = 12.0 Hz,
CH2), 4.02–3.94 (m, 2H, H-3, H-4), 3.86 (m, 3H, H-2, H-6, H-6),
3.70 (m, 1H, H-5), 1.98 (dd, 1H, J = 6.2 Hz, OH).
mannopyranoside (21)
A
mixture of benzyl
a-D-mannopyranoside (20) (7 g,
25.9 mmol) and imidazole (2.72 g, 40 mmol) in DMF (80 mL) was
prepared and cooled to 0 °C. Tert-butyldimethylsilyl chloride
(4.2 g, 28 mmol) was added and the mixture was stirred overnight
while the temperature was allowed to rise to ambient. The reaction
mixture was diluted with water (200 mL) and the product was
extracted with ethyl acetate (3 Â 70 mL). The combined organic
extracts were washed with water and brine, then dried over
Na2SO4. Inorganic salts were filtered off, the solvent was evapo-
rated to dryness, and the product was purified by column chroma-
tography using hexanes–ethyl acetate gradient for elution.
Fractions containing product were combined and evaporated to
dryness. The residual solvents were removed using a high-vacuum
13C NMR (500 MHz, CDCl3): d 138.55, 138.48, 138.28, 137.25,
(C-arom), 128.52, 128.49, 128.44, 128.16, 127.93, 127.88, 127.81,
127.78, 127.71, 127.65, (C-arom), 97.64 (C-1), 80.27 (C-3), 75.33
(CH2), 74.99 (C-2, C-4), 73.01 (CH2), 72.59 (C-5), 72.38 (CH2),
69.20 (CH2), 62.39 (C-6).
4.2.18. Synthesis of benzyl 2,3,4-tri-O-benzyl-6-deutero-a-D-
mannopyranoside (25)
Benzyl 2,3,4-tri-O-benzyl-a-D-mannopyranoside (23) (3.0 g,
5.5 mmol) was oxidized according to the method reported in the
literature19 and, without purification, such obtained crude alde-
hyde (24) was reduced with NaBD4 (general procedure C). After
the reaction was completed, the reaction mixture was diluted with
CH2Cl2 (100 mL) and washed with water until neutral. The crude
product was purified by column chromatography using hexanes–
ethyl acetate gradient for elution. Fractions containing product
were combined and evaporated to dryness. The residual solvents
were removed using a high-vacuum oil pump to give 2.52 g of
oil pump to give 8.5 g of (21) (yield 85%). Mp 68–69 °C, [
(c = 1.7, CHCl3).
a]D +56.3°
1H NMR (300 MHz, DMSO-d6 + D2O):
d 7.40–7.26 (m,5H,
H-arom), 4.69 (d, 1H, J = 1.5 Hz, H-1), 4.66 (d, 1H, J = 11.8 Hz, CH2),
4.42 (d, 1H, J = 11.8 Hz, CH2), 3.92 (dd, 1H, J = 10.9 Hz, J = 1.3 Hz,
H-3), 3.66–3.57 (m, 2H, H-2, H-4), 3.49 (dd, 1H, J = 9.0 Hz,
J = 3.3 Hz, H-6), 3.46–3.40 (m, 1H, H-5), 3.33 (dd, 1H, J = 9.0 Hz,
J = 9.5 Hz, H-6), 0.87 (s, 9H, t-BuSi), 0.06, 0.05 (2s, 3Hea, Me2Si).
(25) as a colorless oil (yield 84%). [a] + 50.1° (c = 1.48, CHCl3);
D
4.2.16. Synthesis of benzyl 2,3,4-tri-O-benzyl-6-tert-
HRMS calcd for C34H35DO6 [M+Na]+ m/z 564.25; found, m/z 564.55.
1H NMR (500 MHz, CDCl3): d 7.37–7.24 (m, 20H, H-arom), 4.94
(d, 1H, J = 10.8 Hz, CH2), 4.91 (br s, 1H, H-1), 4.75 (d, 1H, J = 12.3 Hz,
CH2), 4.69–4.60 (m, 5H, CH2), 4.43 (d, 1H, J = 12.0 Hz, CH2),
4.02–3.94 (m, 2H, H-3, H-4), 3.82 (br s, 1H, H-2), 3.78–3.73 (m,
1H, H-6), 3.72–3.67 (m, 1H, H-5), 1.98 (d, 1H, J = 6.2 Hz, OH).
13C NMR (500 MHz, CDCl3): d 138.67, 138.61, 138.37, 137.37,
(C-arom), 128.60, 128.56, 128.53, 128.24, 128.03, 127.99, 127.97,
127.87, 127.79, 127.73, (C-arom), 97.72 (C-1), 80.36 (C-3), 75.39
(CH2), 75.11 (C-2), 75.04 (C-4), 73.08 (CH2), 72.73 (C-5), 72.44
(CH2), 69.25 (CH2), 62.05 (t, J = 20 Hz, C-6).
butyldimethylsilyl-
a
-
D
-mannopyranoside (22)
-mannopyranoside
The 6-tert-butyldimethylsilyl-
a-
D
(21),
(3.5 g, 9.1 mmol) was dissolved in DMF (50 mL). NaH (60% mineral
oil suspension) (2.15 g, 54 mmol) was added and the mixture was
stirred at room temperature for 10 min, then benzyl bromide
(6.5 mL, 54 mmol) was added. The mixture was heated to 50 °C
for 10 min. The mixture was cooled down to room temperature;
then hexanes (100 mL), followed by acetic acid (1.53 mL,
26.7 mmol) in water (150 mL), were added. After 15 min of vigor-
ous stirring layers were separated. The organic layer was washed
with water, then dried over Na2SO4. Inorganic salts were filtered
off, the solvent was evaporated to dryness and the product was
purified by column chromatography using hexanes–ethyl acetate
gradient for elution. Fractions containing product were combined
and evaporated to dryness. The residual solvents were removed
using a high-vacuum oil pump to give 4.56 g of (22) as a colorless
4.2.19. Synthesis of 6-deutero-D-mannopyranoside (26)
Benzyl 2,3,4-tri-O-benzyl-6-deutero-
a-D-mannopyranoside
(25) (3.4 g, 6.3 mmol) was debenzylated according to general pro-
cedure B). After purification, 0.93 g of (25) (yield 82%) was ob-
tained; mp 122–129 °C, [a] +14.2° (c = 1.4, H2O).
D
oil (yield 77%). [a] +45.0° (c = 1.56, CHCl3).
D
1H NMR (300 MHz, CDCl3): d 7.38–7.24 (m, 20H, H-arom), 4.92
(d, 1H, J = 10.8 Hz, CH2), 4.91 (d, 1H, J = 1.6 Hz, H-1), 4.75–4.60
(m, 6H, CH2), 4.42 (d, 1H, J = 10.8 Hz, CH2), 3.98–3.92 (m, 2H, H-
6, H-6), 3.88–3.83 (m, 2H, H-3, H-4), 3.80 (dd, 1H, J = 1.8 Hz,
J = 2.5 Hz, H-2), 3.67 (ddd, 1H, J = 9.3 Hz, J = 5.8 Hz, J = 2.6 Hz, H-
5), 0.90 (s, 9H, t-BuSi), 0.08, 0.07 (2s, 3Hea, Me2Si).
Acknowledgments
This work was supported by grants from the CERN Foundation
(WP) and Viragh Foundation (WP).
We also acknowledge the NCI Cancer Center Support Grant
CA016672 for the support of the NMR and Pharmacology and Ana-
lytical Facilities at MD Anderson Cancer Center.
4.2.17. Synthesis of benzyl 2,3,4-tri-O-benzyl-a-D-
mannopyranoside (23)
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