Stereoselective synthesis of UDP-2-(2-ketopropyl)galactose aided by di-tert-butylsilylene protecting group

Article abstract of DOI:10.1007/s10719-015-9581-y

UDP-2-(2-ketopropyl)galactose (1) has been utilized as a valuable probe for profiling proteins modified by O-GlcNAc. In this work, we developed a protocol for efficient synthesis of 1. Thus, 2-methallylgalactose derivative 11, a synthetic intermediate for

Full text of DOI:10.1007/s10719-015-9581-y

Glycoconj J
DOI 10.1007/s10719-015-9581-y
ORIGINAL ARTICLE
Stereoselective synthesis of UDP-2-(2-ketopropyl)galactose aided
by di-tert-butylsilylene protecting group
Yasuharu Sakamoto1 & Tsuyoshi Ohta1 & Yukishige Ito1,2
Received: 30 January 2015 /Revised: 5 March 2015 /Accepted: 10 March 2015
#
Springer Science+Business Media New York 2015
Abstract UDP-2-(2-ketopropyl)galactose (1) has been uti-
lized as a valuable probe for profiling proteins modified by
O-GlcNAc. In this work, we developed a protocol for efficient
synthesis of 1. Thus, 2-methallylgalactose derivative 11, a
synthetic intermediate for the compound 1, was prepared by
stereoselective iodination and methallylation at C-2 position,
through exploitation of 4,6-O-di-tert-butylsilylene protecting
group.
O-GlcNAc O-linked β-N-acetylglucosamine
OGT
PEG
Py
O-linked β-N-acetylglucosamine transferase
polyethylene glycol
pyridine
THF
UDP
UMP
tetrahydrofuran
uridine diphosphate
uridine monophosphate
Introduction
.
.
Keywords Stereoselective synthesis Galactose derivative
.
.
Di-tert-butylsilylene Iodination Radical methallylation
Among various types of protein glycosylations, O-linked β-
N-acetylglucosamine (O-GlcNAc) modification (O-
GlcNAcylation) is unique as it occurs on serine or threonine
residues of nuclear and cytoplasmic proteins [1–4]. Introduc-
tion of O-GlcNAc is dynamically regulated by a pair of
counteracting enzymes, O-GlcNAc transferase (OGT) and
O-GlcNAcase (OGA) [5–12]. O-GlcNAcylation, which has
been found on more than 1,000 sites of various proteins, con-
trols transcription, translation, signaling, and cytoskeleton or-
ganization in competition or cooperation with protein phos-
phorylation [3]. Furthermore, it has been attracting growing
attention as a histone code [13–16].
UDP-2-(2-ketopropyl)galactose (1) is utilized as a valuable
tool for facile profiling of proteins modified by O-GlcNAc
[17–19] (Scheme 1). Namely, O-GlcNAc-modified proteins
are chemoenzymatically labeled with UDP-ketogalactose 1
by a β-1,4-galactosyltransferase mutant (Y289L GalT) and
introduced ketogalactose serves as a handle for
chemoselective ligation with aminooxy-functionalized biotin-
[17, 18] or PEG-tag [19].
Abbreviations
AIBN
DMF
DTBS
ESI
α,α′-azobisisobutyronitrile
N,N-dimethylformamide
di-tert-butylsilylene
electrospray ionization
GalT
NIS
β-1,4-galactosyltransferase
N-iodosuccinimide
OGA
O-linked β-N-acetylglucosamine hydrolase
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-015-9581-y) contains supplementary material,
which is available to authorized users.
* Yasuharu Sakamoto
ysakamot@riken.jp
* Yukishige Ito
yukito@riken.jp
1
Previously reported syntheses of 1 [17, 20, 21] employed
iodination of tri-O-acetylgalactal (2) and following radical
methallylation of the resulting iodide 3 as key reactions. The
iodination, however, was reported to afford a mixture of
Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
2
Japan Science and Technology Agency (JST), ERATO Glycotrilogy
Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

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Scheme 1 Reported synthesis of UDP-ketogalactose 1
equatorial (3) and axial (4) iodides. For subsequent transfor-
mation, the minor isomer 3 isolated in 35 % yield was utilized
[21]. In addition, methallylation of 3 was not entirely
stereoselective (5:6=ca. 7:1) [20].
Recently, α-selective glycosylation of galactose and galac-
tosamine derivatives protected with 4,6-O-di-tert-butylsilylene
(DTBS) group was reported [22–24]. In these cases, bulky
DTBS group forces nucleophiles to attack from the convex
side, giving α-glycosides with high selectivity. By analogy,
we anticipated that the iodination and methallylation would
proceed with favorable stereoselectivity when a DTBS group
is installed to galactal, leading to an efficient access to inter-
mediates for 2-(2-ketopropyl)galactose derivatives such as 1.
anomeric composition (α:β=13:1) different from previous
reports.
Methallylation of the iodides was conducted under Keck
conditions [25] based on the reported procedure [11]
(Table 1). As an initial attempt, reaction of the tetraacetate 3
(α:β=13:1) with methallyltributyltin in the presence of AIBN
was conducted in refluxing benzene, which gave a mixture of
isomers composed of the equatorially methallylated product 5
(α:β=1:0.23) and its C-2 epimer 6 (α only) in 62 % yield
(5:6=0.65:1) (Table 1, entry 1). This result was somewhat
unexpected in light of the previous report that the tetraacetate
3 (mainly β) gave the desired 5 as the major product [20]. On
the other hand, methallylation of 9 (α:β=13:1) having the 4,6-
O-DTBS group preferentially gave the equatorial product 11
together with the axial product 12 (11:12=4.6:1) in 89 % yield
(entry 2). In this case, a bulky silylene group exhibited a pos-
itive effect indeed in enhancing a proportion of the desired
isomer. We also observed that the β-anomer of 9 exclusively
gave 11, while the α-anomer afforded an isomeric mixture of
11 and 12 (4.2:1). These results indicated that the configura-
tion of the anomeric acetate affects the stereochemistry of the
methallylation.
With the above knowledge in hand, acetylation of the
hemiacetal 13 was examined to seek for conditions that give
higher proportion of the β-acetate. To prepare 13, iodination
of the galactal 8 was conducted under aqueous conditions,
which gave the equatorial iodide 13 along with 14, which
were used for the following acetylation without separation.
As shown in Table 2, the use of Ac2O and pyridine in CH2Cl2
provided 9 in which the β-anomer was dominant (α:β=1:2.1)
(entry 2), while more reactive AcCl provided larger proportion
Results and discussion
To begin with, the galactal (7) was converted to 4,6-O-DTBS
t
derivative 8 by sequential treatment with Bu2Si(OTf)2 and
Ac2O in pyridine (Scheme 2). Subsequent iodination was car-
ried out by treatment with NIS in the presence of AcOH at
room temperature, which, after acetylation, gave the equato-
rial iodide 9 as a 13:1 mixture of α- and β-anomers in 85 %,
together with the axial isomer 10 which was isolated in 11 %
as an α-anomer. Therefore, the introduction of 4,6-O-DTBS
group into the galactal was shown to exhibit a favorable effect
in controlling the stereochemistry of the C-2 position in an
equatorial selective manner (9:10=7.7:1). Deprotection of
the DTBS group of 9 by using HF•Py in THF and successive
acetylation afforded the known iodide 3 [20, 21], albeit with
Scheme 2 Iodination of 4,6-O-DTBS-protected galactal 8

Glycoconj J
Table 1 Radical methallylation of 2-iodogalactose derivatives
Table 2 Stereoselectivity of acetylation for 1-hydroxy-2-iodogalactose derivative

Glycoconj J
of the α-anomer (entry 1). Other solvents were shown to be
less suitable (entry 3 and 4). Interestingly, acetylation of the
axial iodide 14 afforded only α-anomer 10 under any reaction
conditions.
Experimental
General methods
As expected, methallylation of the iodide 9 now provided
the desired equatorial product 11 as a major (11:12=14.9:1)
(Table 1, entry 3). In addition, the axial iodide 10 was also led
predominantly to 11 (11:12=4.2:1) (entry 4). Noticeably, the
product ratio in the latter case was nearly identical to that
observed for the α-anomer of equatorial iodide 9 (entry 2–
4). These results indicate that no significant anomerization
occurs and anomeric configuration of the acetate is a decisive
factor for stereoselective methallylation.
Having established a stereoselective access to the
methallylated derivative 11, our experimentation was continued
to lead the latter to UDP-2-(2-ketopropyl)galactose (1)
(Scheme 3). Namely, a mixture of the methallylated compounds
11 and 12 (11:12=14.9:1) produced from the iodide 9 was sub-
jected to deprotection of the DTBS group by using HF•Py in
THF, which was followed by acetylation to afford a mixture of
tetraacetates 5 and 6 in 89 % yield. Subsequent conversion to 1
was conducted based on the reported procedure [21]. It was
commenced by partial deacetylation with hydrazine acetate in
DMF, giving the hemiacetal 15, which was readily isolated by
simple chromatographic purification. Subsequent coupling with
dibenzyl N,N-diisopropylphosphoramidite and ozonolysis to
provide the phosphate 17, which was isolated as a single isomer
in high yield. Finally, debenzylation, coupling with the UMP
component, and deprotection of acetyl groups afforded the
aimed compound 1.
All moisture-sensitive reactions were carried out in
dehydrated solvents (Kanto Chemical Co., Inc.) under nitro-
gen atmosphere. Column chromatography was performed on
silica gel 60 N (spherical, neutral, 40–100 μm, Kanto Chem-
ical Co., Inc.). Optical rotations were measured by a JASCO
DIP 370 polarimeter. NMR spectra were recorded on a JEOL
ECX 400 spectrometer (400 MHz for 1H, 100 MHz for 13C,
162 MHz for 31P) in the indicated solvent. Chemical shifts are
reported in ppm related to internal CHCl3 (7.26 ppm) in
CDCl3 or DHO (4.79 ppm) in D2O for 1H, CDCl3
(77.0 ppm) in CDCl3 or native scale in D2O for 13C, and
external H3PO4 (0.00 ppm) in D2O for 31P. ESI mass spectra
were recorded on a Waters SYNAPT G2 mass spectrometer.
3-O-Acetyl-1,5-anhydro-4,6-O-di-tert-butylsilylene-2-
deoxy-D-lyxo-hex-1-enitol (8)
To a solution of D-galactal (7) (1.36 g, 9.31 mmol) in dry
pyridine (279 mL) was added di-tert-butylsilyl
bis(trifluoromethanesulfonate) (3.4 mL, 10.4 mmol) at room
temperature. After being stirred for 3 h at the same tempera-
ture, acetic anhydride (2.7 mL, 28.6 mmol) was added to the
reaction mixture at room temperature. After being stirred for
1 h at the same temperature, the reaction mixture was concen-
trated in vacuo. The residue was dissolved in EtOAc, washed
with ice-cooled 1 N HCl solution, saturated NaHCO3 solution,
brine, and dried over MgSO4. After filtration and removal of
the solvent in vacuo, the residue was purified by column chro-
matography on silica gel (hexane/EtOAc=30/1) to give 8
In conclusion, we developed a renewed procedure to pre-
pare UDP-2-(2-ketopropyl)galactose (1), through exploitation
of 4,6-O-DTBS protecting group as an α-directing element.
The present procedure gave UDP-2-(2-ketopropyl)galactose
(1) in 22 % overall yield. Furthermore, it was revealed that
configuration of anomeric acetate affected stereoselectivity of
the radical methallylation at C-2 position.
(2.28 g, 6.94 mmol, 75 %). [α]23 +132 (c 1.00, MeOH);
D
1H NMR (400 MHz, CDCl3): δ 6.42 (dd, J=6.3, 1.8 Hz,
1H, H-1), 5.25 (m, 1H, H-3), 4.81 (brd, J=4.5 Hz, 1H, H-4),
4.65 (dt, J=6.3, 1.8 Hz, 1H, H-2), 4.27 (dd, J=12.6, 1.8 Hz,
Scheme 3 Synthesis of UDP-ketogalactose 1

Glycoconj J
1H, H-6), 4.23 (dd, J=12.6, 1.8 Hz, 1H, H-6), 3.87 (brs, 1H,
5.31 (dd, J=11.7, 2.7 Hz, 1H, H-3), 4.60 (m, 1H, H-4), 4.59
(dd, J=11.7, 3.2 Hz, 1H, H-2), 4.19 (dd, J=12.8, 2.3 Hz, 1H,
H-6), 4.14 (dd, J=12.8, 1.6 Hz, 1H, H-6), 3.92 (brs, 1H, H-5),
2.17 (s, 3H, Ac), 2.14 (s, 3H, Ac), 1.03 (s, 9H, tBu), 1.00 (s,
9H, tBu); 13C NMR (100 MHz, CDCl3): δ 170.3, 168.9, 92.8,
73.0, 70.7, 69.2, 66.7, 27.5, 27.2, 23.2, 23.1, 20.9, 20.8, 20.7;
β-anomer: 1H NMR (400 MHz, CDCl3): δ 5.86 (d, J=9.4 Hz,
1H, H-1), 4.87 (dd, J=11.6, 3.1 Hz, 1H, H-3), 4.48 (m, 1H,
H-4), 4.32 (dd, J=11.6, 9.4 Hz, 1H, H-2), 4.17–4.26 (m, 2H,
H-6), 3.64 (m, 1H, H-5), 2.18 (s, 3H, Ac), 2.15 (s, 3H, Ac),
H-5), 2.10 (s, 3H, Ac), 1.02 (s, 9H, tBu), 1.01 (s, 9H, tBu); 13
C
NMR (100 MHz, CDCl3): δ 171.0, 145.7, 98.4, 73.1, 67.6,
67.3, 65.0, 27.6, 26.9, 23.3, 20.9, 20.8; HR-ESI-MS: [M+
Na]+ m/z calcd for C16H28O5SiNa 351.1604, found 351.1595.
1,3-Di-O-acetyl-4,6-O-di-tert-butylsilylene-2-deoxy-
2-iodo-D-galactopyranose (9, 10)
Method A using acetic acid as a nucleophile
t
t
1.04 (s, 9H, Bu), 1.00 (s, 9H, Bu); 13C NMR (100 MHz,
To a solution of 8 (1.44 g, 4.38 mmol) and acetic acid (1.3 mL,
22.7 mmol) in dry CH2Cl2 (88 mL) was added N-
iodosuccinimide (1.49 g, 6.62 mmol) at room temperature.
After being stirred for 2 h at the same temperature, a mixture
of 1 M Na2S2O3 solution and saturated NaHCO3 solution was
added to the reaction mixture at room temperature. After being
stirred for 5 min at the same temperature, the organic layer was
separated and washed with a mixture of 1 M Na2S2O3 solution
and saturated NaHCO3 solution, brine, and dried over
MgSO4. After filtration and removal of the solvent in vacuo,
the residue was dissolved in pyridine (26 mL). Acetic anhy-
dride (26 mL) was added to this solution at room temperature.
After being stirred for 63 h at the same temperature, the reac-
tion mixture was concentrated in vacuo azeotropically with
toluene. The residue was purified by column chromatography
on silica gel (hexane/EtOAc=10/1 for 9 then 2/1 for 10) to
give a mixture of 9 (1.92 g, 3.73 mmol, 85 %, α:β=13:1) and
10 (0.260 g, 0.505 mmol, 11 %).
CDCl3): δ 170.1, 169.0, 94.5, 76.7, 72.3, 70.1, 66.5, 27.5,
27.3, 25.9, 23.2, 20.8 (two carbons), 20.7. 10: [α]27 +92.6
D
1
(c 1.03, MeOH); H NMR (400 MHz, CDCl3): δ 6.59 (brs,
1H, H-1), 4.73 (m, 1H, H-4), 4.62 (dd, J=5.4, 3.6 Hz, 1H,
H-3), 4.30 (dd, J=13.0, 3.6 Hz, 1H, H-6), 4.29 (m, 1H, H-2),
4.21 (dd, J=13.0, 1.8 Hz, 1H, H-6), 3.97 (m, 1H, H-5), 2.20
(s, 3H, Ac), 2.09 (s, 3H, Ac), 1.13 (s, 9H, tBu), 1.03 (s, 9H,
tBu); 13C NMR (100 MHz, CDCl3): δ 170.3, 168.4, 97.3,
69.4, 68.0, 67.1, 66.9, 27.9, 27.1, 23.2, 21.1, 20.93, 20.86,
17.5; HR-ESI-MS: [M+Na]+ m/z calcd for C18H31O7ISiNa
537.0781, found 537.0781.
General procedure for methallylation of 2-iodo-sugars
(9 and 10)
A solution of 9 or 10 and methallyltri-n-butyltin (12 eq) in
benzene (10 mL/mmol) bubbled with nitrogen gas for 30 min.
To the resulting solution was added AIBN (0.2 eq) at room
temperature. After being stirred for 80 min under reflux, the
reaction mixture was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (hexane/
EtOAc=15/1) to give 2-methallylated products (11α, 11β,
12α). HR-ESI-MS: [M+Na]+ m/z calcd for C22H38O7SiNa
Method B using water as a nucleophile
To a solution of 8 (1.09 g, 3.32 mmol) and H2O (3.3 mL) in
CH2Cl2 (66 mL) was added N-iodosuccinimide (2.24 g,
9.96 mmol) at room temperature. After being stirred for 3 h
at the same temperature, a mixture of 1 M Na2S2O3 solution
and saturated NaHCO3 solution was added to the reaction
mixture at room temperature. After being stirred for 5 min at
the same temperature, the organic layer was separated and
washed with a mixture of 1 M Na2S2O3 solution and saturated
NaHCO3 solution, brine, and dried over MgSO4. After filtra-
tion and removal of the solvent in vacuo, the residue was
dissolved in CH2Cl2 (16 mL). To this solution was added
pyridine (1.62 mL, 20.0 mmol) and acetic anhydride
(0.95 mL, 10.1 mmol) at room temperature. After being stirred
for 20 h at the same temperature, the reaction mixture was
concentrated in vacuo azeotropically with toluene. The resi-
due was purified by column chromatography on silica gel
(hexane/EtOAc=10/1 for 9 then 1/1 for 10) to give 9
(1.40 g, 2.72 mmol, 82 %, α:β=1:2.1) and 10 (0.217 g,
0.422 mmol, 13 %). 9: HR-ESI-MS: [M+Na]+ m/z calcd for
1
465.2284, found 465.2275; 11α: H NMR (400 MHz,
CDCl3): δ 6.06 (d, J=3.6 Hz, 1H, H-1), 4.92 (dd, J=11.7,
2.7 Hz, 1H, H-3), 4.77 (brs, 1H, vinyl), 4.61 (brs, 1H, vinyl),
4.56 (brd, J=2.7 Hz, 1H, H-4), 4.20 (dd, J=12.6, 2.2 Hz, 1H,
H-6), 4.14 (dd, J=12.6, 1.8 Hz, 1H, H-6), 3.75 (brs, 1H, H-5),
2.80 (dddd, J=11.7, 10.8, 4.9, 3.6 Hz, 1H, H-2), 2.21 (dd, J=
13.9, 4.9 Hz, 1H, allyl), 2.11 (s, 3H, Ac), 2.09 (s, 3H, Ac),
1.92 (dd, J=13.9, 10.8 Hz, 1H, allyl), 1.73 (brs, 3H, Me), 1.04
(s, 9H, tBu), 1.01 (s, 9H, tBu); 13C NMR (100 MHz, CDCl3):
δ 170.9, 169.4, 141.7, 112.9, 92.8, 72.6, 69.2, 69.1, 67.0, 35.3,
33.7, 27.5, 27.2, 23.2, 21.7, 20.88, 20.86, 20.7. 11β: 1H NMR
(400 MHz, CDCl3): δ 5.50 (d, J=9.0 Hz, 1H, H-1), 4.67 (brs,
1H, vinyl), 4.61 (brs, 1H, vinyl), 4.58 (dd, J=11.2, 2.7 Hz, 1H,
H-3), 4.47 (brd, J=2.7 Hz, 1H, H-4), 4.16–4.23 (m, 2H, H-6),
3.50 (m, 1H, H-5), 2.60 (m, 1H, H-2), 2.01–2.21 (m, 2H,
allyl), 2.08 (s, 3H, Ac), 2.05 (s, 3H, Ac), 1.76 (brs, 3H, Me),
t
t
1.06 (s, 9H, Bu), 1.00 (s, 9H, Bu); 13C NMR (100 MHz,
CDCl3): δ 170.6, 169.4, 143.4, 111.6, 94.9, 75.1, 71.5, 68.5,
66.9, 36.4, 35.7, 27.4, 27.3, 23.2, 21.9, 20.9, 20.72, 20.66.
1
C18H31O7ISiNa 537.0781, found 537.0779; α-anomer: H
NMR (400 MHz, CDCl3): δ 6.41 (d, J=3.2 Hz, 1H, H-1),

Glycoconj J
12α: 1H NMR (400 MHz, CDCl3): δ 6.18 (brs, 1H, H-1), 5.12
(dd, J=5.8, 3.1 Hz, 1H, H-3), 4.83 (brs, 1H, vinyl), 4.76 (brs,
1H, vinyl), 4.63 (brd, J=3.1 Hz, 1H, H-4), 4.29 (dd, J=13.0,
3.6 Hz, 1H, H-6), 4.19 (dd, J=13.0, 1.3 Hz, 1H, H-6), 3.88 (m,
1H, H-5), 2.69 (dd, J=15.2, 11.2 Hz, 1H, allyl), 2.50 (brd, J=
15.2 Hz, 1H, allyl), 2.14 (s, 3H, Ac), 2.10–2.18 (m, 1H, H-2),
2.07 (s, 3H, Ac), 1.71 (brs, 3H, Me), 1.07 (s, 9H, tBu), 1.02 (s,
9H, tBu); 13C NMR (100 MHz, CDCl3) δ 170.5, 169.0, 143.3,
112.1, 94.8, 69.6, 69.1, 68.7, 67.3, 37.2, 34.4, 27.7, 27.2, 23.1,
22.2, 21.1, 21.0, 20.6.
(400 MHz, CDCl3): δ 6.13 (brs, 1H, H-1), 5.36 (dd, J=5.8,
3.6 Hz, 1H, H-3), 5.34 (m, 1H, H-4), 4.85 (brs, 1H, vinyl),
4.79 (brs, 1H, vinyl), 4.29 (dt, J=1.8, 6.7 Hz, 1H, H-5), 4.15
(dd, J=11.2, 6.7 Hz, 1H, H-6), 4.08 (dd, J=11.2, 6.7 Hz, 1H,
H-6), 2.43 (dd, J=14.8, 11.2 Hz, 1H, allyl), 2.32 (brd, J=
14.8 Hz, 1H, allyl), 2.11–2.20 (m, 1H, H-2), 2.11 (s, 6H,
two Acs), 2.033 (s, 3H, Ac), 2.026 (s, 3H, Ac), 1.70 (brs,
3H, Me); 13C NMR (100 MHz, CDCl3): δ 170.5, 170.0,
169.9, 168.9, 142.5, 113.2, 93.7, 68.5, 66.6, 66.3, 61.7,
37.7, 34.2, 22.0, 21.1, 20.79, 20.72, 20.67.
1,3,4,6-Tetra-O-acetyl-2-deoxy-
3,4,6-Tri-O-acetyl-2-deoxy-2-methallyl-D-galactose (15)
2-methallyl-D-galactopyranose (5, 6)
To a solution of the mixture of 5α, 5β, and 6α (3.4:10.7:1;
0.201 g, 0.520 mmol) in dry DMF (1.8 mL) was added a
powdered hydrazine acetate (95.8 mg, 1.04 mmol) at room
temperature. During being stirred for 4 h at the same temper-
ature, hydrazine acetate (50.2 mg, 0.545 mmol) in DMF
(2 mL) was hourly added to the reaction mixture three times.
The reaction mixture was diluted with EtOAc (60 mL) and
washed with brine (40 mL). The aqueous layer was extracted
with EtOAc three times and the combined organic layer was
washed with brine and dried over MgSO4. After filtration and
removal of the solvent in vacuo, the residue was purified by
column chromatography on silica gel (hexane/EtOAc=3/1) to
A mixture of 11α, 11β, and 12α (3.8:11.1:1; 0.835 g,
1.89 mmol) in dry THF (20 mL) was added hydrogen fluoride
pyridine (0.9 mL) at room temperature. After being stirred for
70 min at the same temperature, EtOAc (50 mL) and saturated
NaHCO3 solution (50 mL) was added to the reaction mixture
at room temperature. After being stirred for 5 min at the same
temperature, the reaction mixture was extracted with EtOAc
(150 mL). The aqueous layer was further extracted with
EtOAc (50 mL) three times. The combined organic layer
was dried over MgSO4. After filtration and removal of the
solvent in vacuo, the residue was dissolved in pyridine
(12 mL). Acetic anhydride (12 mL) was added to this solution
at room temperature. After being stirred for 16 h at the same
temperature, the reaction mixture was concentrated in vacuo
azeotropically with toluene. The residue was purified by col-
umn chromatography on silica gel (hexane/EtOAc=9/2) to
give 0.651 g (89 %) of corresponding tetraacetates
(5α:5β:6α=3.4:10.7:1). HR-ESI-MS: [M+Na]+ m/z calcd
give 15 (0.163 g, 0.473 mmol, 91 %, α:β=9:1 in CDCl3).
1
[α]22 +85.2 (c 1.01, CHCl3); H NMR (400 MHz, CDCl3)
D
α-anomer: δ 5.34 (dd, J=3.1, 1.3 Hz, 1H, H-4), 5.26 (d, J=
3.1 Hz, 1H, H-1), 5.16 (dd, J=11.7, 3.1 Hz, 1H, H-3), 4.80
(brs, 1H, vinyl), 4.78 (brs, 1H, vinyl), 4.43 (ddd, J=6.7, 6.3,
1.3 Hz, 1H, H-5), 4.11 (dd, J=11.2, 6.3 Hz, 1H, H-6), 4.07
(dd, J=11.2, 6.7 Hz, 1H, H-6), 2.39 (dddd, J=11.7, 9.4, 5.4,
3.1 Hz, 1H, H-2), 2.15 (s, 3H, Ac), 2.09–2.17 (m, 2H, allyl),
2.05 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.75 (brs, 3H, Me); β-
anomer: δ 5.25 (m, 1H, H-4), 4.84 (dd, J=11.2, 3.2 Hz, 1H,
H-3), 4.75 (brs, 1H, vinyl), 4.71 (brs, 1H, vinyl), 4.64 (d, J=
8.5 Hz, 1H, H-1), 4.15 (dd, J=11.7, 6.3 Hz, 1H, H-6), 4.12
(dd, J=11.7, 6.7 Hz, 1H, H-6), 3.88 (ddd, J=6.7, 6.3, 0.9 Hz,
1H, H-5), 2.20 (m, 1H, H-2), 2.11 (s, 3H, Ac), 2.09–2.17 (m,
2H, allyl), 2.05 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.77 (brs, 3H,
Me); 13C NMR (100 MHz, CDCl3) α-anomer: δ 170.6,
170.39, 170.38, 142.2, 112.5, 93.2, 69.5, 67.1, 66.4, 62.4,
36.4, 35.3, 22.3, 20.73, 20.71, 20.70; β-anomer: δ 170.6,
170.4, 170.2, 143.5, 112.2, 98.0, 72.1, 70.7, 66.1, 62.0,
40.3, 36.3, 22.6, 20.69, 20.67, 20.5; HR-ESI-MS: [M+Na]+
m/z calcd for C16H24O8Na 367.1369, found 367.1360.
1
for C18H26O9Na 409.1475, found 409.1469; 5α: H NMR
(400 MHz, CDCl3): δ 6.07 (d, J=3.1 Hz, 1H, H-1), 5.34 (m,
1H, H-4), 5.12 (dd, J=11.7, 3.1 Hz, 1H, H-3), 4.77 (brs, 1H,
vinyl), 4.61 (brs, 1H, vinyl), 4.21 (ddd, J=7.2, 6.7, 0.9 Hz,
1H, H-5), 4.09 (dd, J=11.7, 7.2 Hz, 1H, H-6), 4.04 (dd, J=
11.7, 6.7 Hz, 1H, H-6), 2.53 (m, 1H, H-2), 2.15 (s, 3H, Ac),
2.12 (s, 3H, Ac), 2.10–2.20 (m, 1H, allyl), 2.01 (s, 3H, Ac),
1.99 (s, 3H, Ac), 1.91 (dd, J=13.9, 10.3 Hz, 1H, allyl), 1.72
(brs, 3H, Me); 13C NMR (100 MHz, CDCl3): δ 170.44,
170.35, 170.27, 169.0, 141.1, 113.3, 92.0, 69.6, 68.4, 66.2,
61.5, 35.3, 35.2, 21.9, 20.8, 20.70, 20.68, 20.65; 5β: 1H NMR
(400 MHz, CDCl3): δ 5.55 (d, J=9.4 Hz, 1H, H-1), 5.27 (dd,
J=3.1, 0.9 Hz, 1H, H-4), 4.84 (dd, J=11.7, 3.1 Hz, 1H, H-3),
4.67 (brs, 1H, vinyl), 4.61 (brs, 1H, vinyl), 4.14 (dd, J=11.2,
7.2 Hz, 1H, H-6), 4.09 (dd, J=11.2, 6.7 Hz, 1H, H-6), 3.95
(ddd, J=7.2, 6.7, 0.9 Hz, 1H, H-5), 2.40 (m, 1H, H-2), 2.14 (s,
3H, Ac), 2.10–2.19 (m, 1H, allyl), 2.08 (s, 3H, Ac), 2.02 (s,
3H, Ac), 2.00–2.09 (m, 1H, allyl), 1.97 (s, 3H, Ac), 1.73 (brs,
3H, Me); 13C NMR (100 MHz, CDCl3): δ 170.4, 170.3,
170.0, 168.9, 142.7, 112.2, 94.7, 72.0, 71.4, 65.6, 61.4,
Dibenzyl (2-acetonyl-2-deoxy-3,4,6-tri-O-
acetyl-α-D-galactopyranosyl) phosphate (17)
To a solution of 15 (77.4 mg, 0.225 mmol) and 1H-tetrazole
(63.0 mg, 0.899 mmol) in dry CH2Cl2 (1.6 mL) was added
dibenzyl N,N-diisopropylphosphoramidite (190 μL,
1
37.3, 36.4, 21.9, 20.8, 20.70, 20.65, 20.5; 6α: H NMR

Glycoconj J
0.571 mmol) at −30 °C. After the reaction mixture was slowly
warmed up to room temperature for 3 h, dibenzyl N,N-
diisopropylphosphoramidite (38 μL, 0.11 mmol) was added
to the reaction mixture at room temperature. After being
stirred for 1 h at the same temperature, the reaction mixture
was diluted with Et2O (30 mL), washed with cold brine twice,
and dried over MgSO4. After filtration and removal of the
solvent in vacuo, the residue was dissolved in dry CH2Cl2
(1.8 mL). Ozone was bubbled into this solution at −78 °C until
a blue color became obvious (2 min). Oxygen was then bub-
bled into the reaction mixture. After the blue color disap-
peared, dimethylsulfide (1.3 mL) was added to the reaction
mixture at −78 °C. After being stirred overnight at room tem-
perature, the reaction mixture was concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(hexane/EtOAc=1/1) to give 17 (0.132 g, 0.218 mmol, 97 %).
1H NMR (400 MHz, CDCl3): δ 7.30–7.40 (m, 10H, Ph), 5.87
(dd, J=6.3, 2.9 Hz, 1H, H-1), 5.31 (m, 1H, H-4), 4.98–5.12
(m, 4H, benzyl), 4.95 (dd, J=12.1, 3.1 Hz, 1H, H-3), 4.27
(brdd, J=6.7, 6.3 Hz, 1H, H-5), 4.06 (dd, J=11.2, 6.3 Hz,
1H, H-6), 3.95 (dd, J=11.2, 6.7 Hz, 1H, H-6), 2.87 (m, 1H,
H-2), 2.38 (dd, J=18.4, 8.1 Hz, 1H, acetonyl), 2.36 (dd, J=
18.4, 5.4 Hz, 1H, acetonyl), 2.12 (s, 3H), 1.97 (s, 3H), 1.93 (s,
3H), 1.89 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 205.9,
170.24, 170.17, 170.0, 135.4 (d, JCP=6.6 Hz), 135.3 (d,
Pak® C18 cartridge with 0.1 M NH4HCO3 solution, gel per-
meation column chromatography on Sephadex G-15 with
0.1 M NH4HCO3 solution and then lyophilization to give 1
(69.9 mg, 0.116 mmol, 48 %). After extensive purification by
HPLC using ODS column (Mightysil RP-18, 20×250 mm,
5 μm) with 0.1 M NH4HCO3 solution, 40.5 mg of 1 was
1
provided. H NMR (400 MHz, D2O): δ 7.94 (d, J=8.1 Hz,
1H, H-6″), 5.95 (d, J=2.7 Hz, 1H, H-1′), 5.94 (d, J=8.1 Hz,
1H, H-5″), 5.55 (dd, J=7.2, 3.1 Hz, 1H, H-1), 4.31–4.37 (m,
2H, H-2′,3′), 4.25 (m, 1H, H-4′), 4.21 (ddd, J=11.7, 4.5,
2.2 Hz, 1H, H-5′), 4.15 (ddd, J=11.7, 5.4, 3.1 Hz, 1H, H-5′),
4.12 (brdd, J=6.7, 5.2 Hz, 1H, H-5), 3.86 (brd, J=3.1 Hz, 1H,
H-4), 3.75 (dd, J=11.2, 3.1 Hz, 1H, H-3), 3.73 (dd, J=11.7,
6.7 Hz, 1H, H-6), 3.68 (dd, J=11.7, 5.2 Hz, 1H, H-6), 2.79
(dd, J=17.9, 5.8 Hz, 1H, acetonyl), 2.73 (dd, J=17.9, 7.2 Hz,
1H, acetonyl), 2.50 (m, 1H, H-2), 2.23 (s, 3H, acetonyl); 13
C
NMR (100 MHz, D2O): δ 214.3, 166.3, 151.8, 141.7, 102.6,
96.2 (d, JCP=6.6 Hz), 88.5, 83.2 (d, JCP=9.4 Hz), 73.8, 71.9,
69.6, 68.2, 68.0, 64.9 (d, JCP=5.6 Hz), 61.4, 41.2, 36.8 (d,
J
CP=8.5 Hz), 29.9; 31P NMR (162 MHz, D2O) δ –11.42 (brd,
J=20.1 Hz), −12.73 (brdd, J=20.1, 5.3 Hz); HR-ESI-MS:
[M+Na]+ m/z calcd for C18H28N2O17P2Na 629.0761, found
629.0750.
Acknowledgments We thank Dr. Yayoi Hongo and Dr. Takemichi
Nakamura for ESI mass measurements. We also thank Ms. Akemi
Takahashi and Ms. Satoko Shirahata for technical assistance. This re-
search was supported by the Advanced Research for Medical Products
Mining Program (09–01) of the National Institute of Biomedical Innova-
tion, Osaka, Japan (NIBIO).
J
CP=6.6 Hz), 128.7, 128.63, 128.60, 128.0, 97.8 (d, JCP=
6.6 Hz), 69.6 (d, JCP=5.6 Hz), 69.5 (d, JCP=5.6 Hz), 68.3,
68.0, 65.9, 61.7, 39.6, 34.3 (d, JCP=7.5 Hz), 29.9, 20.6 (two
carbons), 20.5.
Uridine diphosphate 2-deoxy-2-(2-oxopropyl)
-α-D-galactopyranose (1)
References
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Celite® and concentrated in vacuo. The residue was dissolved
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dicyclohexylcarboxamidine salt (0.252 g, 0.367 mmol), acti-
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was diluted with H2O (20 mL), washed with CH2Cl2 (30 mL)
twice. The aqueous layer was lyophilized and the residue was
purified by short-path column chromatography using Sep-
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