Improved synthesis of UDP-2-(2-ketopropyl)galactose and a first synthesis of UDP-2-(2-ketopropyl)glucose for the site-specific linking of biomolecules via modified glycan residues using glycosyltransferases

Article abstract of DOI:10.1016/j.tet.2011.01.081

The potential of wild-type and mutant glycosyltransferases to produce glycoconjugates carrying sugar moieties with chemical handles has made it possible to conjugate biomolecules with orthogonal reacting groups at specific sites. The synthesis of UDP-2-(2

Full text of DOI:10.1016/j.tet.2011.01.081

Tetrahedron 67 (2011) 2013e2017
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Improved synthesis of UDP-2-(2-ketopropyl)galactose and a first synthesis of
UDP-2-(2-ketopropyl)glucose for the site-specific linking of biomolecules via
modified glycan residues using glycosyltransferases
a
,
b
a
a
*
ꢀ
Andres E. Dulcey , Pradman K. Qasba , Jeffrey Lamb , Gary L. Griffiths
a Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, USA
b Structural Glycobiology Section, Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The potential of wild-type and mutant glycosyltransferases to produce glycoconjugates carrying sugar
moieties with chemical handles has made it possible to conjugate biomolecules with orthogonal reacting
groups at specific sites. The synthesis of UDP-2-(2-ketopropyl)galactose has been previously carried out,
albeit with difficulty and low efficiency. A modified approach has been developed for the synthesis of
UDP-2-(2-ketopropyl)glucose and UDP-2-(2-ketopropyl)galactose, allowing better access to the desired
test compounds, the UDP-2-(2-ketopropyl)glucose and UDP-2-(2-ketopropyl)galactose analogs were
synthesized in eight steps and 4.8% and 5.3% overall yield, respectively, an improvement over the first
generation synthesis involving eight steps and an overall yield of 0.7%.
Received 10 December 2010
Received in revised form 25 January 2011
Accepted 26 January 2011
Available online 2 February 2011
Keywords:
Carbohydrate chemistry
Organic synthesis
Modified sugars
Glycosyltransferases
Published by Elsevier Ltd.
1. Introduction
assembly of novel bio-nanoparticles useful for targeted drug de-
livery and as MRI contrast agents.6,7
Structural information on glycosyltransferases has revealed that
the specificity of these enzymes can be broadened to include mod-
ified sugars with chemical handles that can then be utilized for site-
specific conjugation chemistry. As such, this approach represents
a powerful general method for the site-specific modification of
glycoproteins. Substitution of Tyr289 to Leu in the catalytic pocket of
The synthesis of UDP-2-(2-ketopropyl)glucose (Fig. 1, 8-Glc) has
not been previously reported, while the synthesis of UDP-2-(2-
ketopropyl)galactose (Fig. 1, 8-Gal) was reported in low yields. The
O
N
OH
O
bovine
b-1,4-galactosyltransferase generates a novel glycosyl-
HO
HO
NH
transferase, which can transfer not only galactose, but also modified
galactosyl moieties, such as GalNAc1 and C2-modified galactose
containing various chemical handles, from the corresponding uri-
dine diphosphate (UDP) derivatives.2,3 Similarly, the wild-type
N-acetylgalactosaminyltransferase, which naturally transfers Gal-
NAc from UDP/GalNAc, can also transfer C2-modified galactose units
from their UDP-derivative to the Ser/Thr residue of a polypeptide
acceptor substrate tagged as a fusionpeptide to a non-glycoprotein.4
The potential of wild-type and mutant glycosyltransferases to pro-
duce glycoconjugates carrying sugar moieties with chemical han-
dles has made it possible to conjugate biomolecules with orthogonal
reacting groups at specific sites.5 This methodology has enabled the
O
P
O
O P O
O-
O
O
O
O
O
O-
8-Glc
HO
OH
O
OH
HO
OH
O
NH
O
O
O P O P
O
N
O
O
O- O-
8-Gal
HO
OH
* Corresponding author. Tel.: þ1 301 217 5759; fax: þ1 301 217 5779; e-mail
address: dulceyan@mail.nih.gov (A.E. Dulcey).
Fig. 1. UDP-2-(2-ketopropyl)glucose 8-Glc and UDP-2-(2-ketopropyl)galactose 8-Gal.
0040-4020/$ e see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2011.01.081

2014
A.E. Dulcey et al. / Tetrahedron 67 (2011) 2013e2017
lack of efficient syntheses of these substrates represents a roadblock
tothe continuing development of this promising technology, as both
are required for the site-specific conjugation of biomolecules via
glycan residues using glycosyltransferases. The linking technique
enables conjugation of the glycoprotein-appended 2-(2-ketopropyl)
sugar to aminooxy derivatives, such as aminooxybiotin and has wide
potential for future conjugation of cytotoxic drugs, cytokines,
radionuclides for imaging and radioimmunotherapy, and lipids for
the assembly of liposomes for targeted drug delivery.6,7
5-Glc and 5-Gal were not isolated. After a quick workup, 5-Glc and
5-Gal were subjected to ozonolysis at ꢀ78 ꢁC followed by the ad-
dition of dimethyl sulfide to obtain
6-Glc in 68% yield from 4-Glc, and
6-Glc in 60% yield from 4-Gal.
a-D-glucopyranoside phosphate
a-D-glucopyranoside phosphate
Hydrogenolysis of the benzyl groups in 6-Glc and 6-Gal, fol-
lowed by condensation with UMP-morpholidate and deprotection
of the acetyl groups afforded the target UDP-2-(2-ketopropyl)glu-
cose (8-Glc) in 49% yield from 6-Glc, and UDP-2-(2-ketopropyl)
galactose (8-Gal) in 53% yield from 6-Gal.
A major obstacle was presented by the sequence illustrated in
Scheme 2, employed in the first generation synthesis.2 The se-
quence was troubled by low-yielding transformations and un-
wanted byproduct formation due to the instability of the
intermediates, along with a need to perform two oxidation steps.
The yield reported in the first generation synthesis from in-
termediate 3-Gal to intermediate 6-Gal was of only 26%. These
shortcomings would prove detrimental if the synthesis was to be
scaled up eventually, as requested. The new approach shown in
Scheme 1 applies redox economy in organic synthesis,10 and illus-
trates how unwanted byproduct formation (Aldol condensation)
was avoided by performing just one combined oxidation step11
after the phosphoramidite coupling had taken place (4 to 5 to 6
in Scheme 1). This aided in scale up and afforded increased yields
for key intermediates 6-Gal and 6-Glc with the yields from in-
termediate 3 to intermediate 6 now 42% in the galactose series and
54% in the glucose series (Scheme 1).
2. Results and discussion
‘2-(2-Ketopropyl)sugars’, which are C2-carbon isosteres of 2-N-
acetamidosugars, were first introduced by Bertozzi8 as novel ana-
logs possessing a ketone group for chemoselective reaction with
aminooxy- or hydrazido-reagents. The first generation synthesis of
UDP-2-(2-ketopropyl)galactose (8-Gal) was carried out by Hsieh-
Wilson’s group,2 albeit with difficulty and low efficiency. The
modified approach for the synthesis of UDP-2-(2-ketopropyl)glu-
cose (8-Glc) and UDP-2-(2-ketopropyl)galactose (8-Gal), developed
at the Imaging Probe Development Center is displayed in Scheme 1.
Thus, from commercially available tri-O-acetyl-
D-glucal (1-Glc)
or tri-O-acetyl- -galactal (1-Gal), equatorial iodination was effec-
D
ted with N-iodosuccinimide (NIS) and acetic acid in dry dichloro-
methane to afford equatorial iodosugars 2-Glc and 2-Gal in 25% and
35% yield, respectively, with an a:b ratio of w1:10 for the Glc series
and w1:2 for the Gal series. Keck radical allylation9 with meth-
allyltributylstannane and catalytic AIBN (2,20-azobis(2-methyl-
propionitrile)) afforded the desired equatorial methallyl
compounds 3-Glc and 3-Gal in 70% and 68% yield, respectively,
along with <5% of the axial epimer, separable chromatographically.
Anomeric deprotection was effected with hydrazine acetate in
DMF, obtaining lactol 4-Glc and 4-Gal, which was coupled with
dibenzyl N,N-diisopropylphosphoramidite at ꢀ30 ꢁC. Compounds
3. Conclusion
The modified approach developed for the synthesis of UDP-2-
(2-ketopropyl)glucose and UDP-2-(2-ketopropyl)galactose, herein
described in full experimental detail, has allowed much improved
access to the desired test substrates. The UDP-2-(2-ketopropyl)
Bu3Sn
b)
OAc
x
H2NNH2.HOAc
DMF
70% Gal
83% Glc
OAc
x
NIS, AcOH
CH2Cl2
OAc
x
O
OAc
a)
c)
AIBN
O
y
O
y
O
y
OH
x
y
OAc
AcO
OAc
AcO
35% eq Gal
25% eq Glc
AcO
68% Gal
70% Glc
I
OAc
D-Galactal 1-Gal x = OAc, y = H
D-Glucal 1-Glc x = H, y = OAc
2-Gal x = OAc, y = H
2-Glc x = H, y = OAc
3-Gal x = OAc, y = H
3-Glc x = H, y = OAc
4-Gal x = OAc, y = H
4-Glc x = H, y = OAc
x
(BnO)2PNiPr2
OAc
1. 10% Pd/C, H2
2. UMP-Morpholidate
1H-Tetrazole
i) O3, -78 o
ii) Me2S
C
x
f)
OAc
d)
e)
1H-Tetrazole
O
y
O
y
-30 oC to RT
AcO
O
P
AcO
OBn
OBn
O
OBn
60% from 4-Gal
68% from 4-Glc
O
O
P
OBn
6-Gal x = OAc, y = H
6-Glc x = H, y = OAc
5-Gal x = OAc, y = H
5-Glc x = H, y = OAc
x
O
OAc
O
y
NH
O
AcO
O
O
P
MeOH, H2O
Et3N
g)
O
P
O
O
N
O
O
O-
O-
53% from 6-Gal
49% from 6-Glc
HO
OH
7-Gal x = OAc, y = H
7-Glc x = H, y = OAc
8-Gal x = OAc, y = H
8-Glc x = H, y = OAc
Scheme 1. Reagents and conditions. (a) N-Iodosuccinimide (1.5 equiv), AcOH (2 equiv) CH2Cl2, rt, 45 min. (b) Methallyltributylstannane (2 equiv), AIBN (0.15 equiv), benzene, reflux,
3 h. (c) Hydrazine acetate (1.2 equiv), DMF, rt, 2 h. (d) 1-H-Tetrazole (4 equiv), dibenzyl N,N-diisopropylphosphoramidite (2.5 equiv), CH2Cl2, ꢀ30 ꢁC to rt 3 h. (e) (i) Ozone,
CH2Cl2eCH3OH, ꢀ78 ꢁC, 40 min. Then (ii) Me2S (excess), ꢀ78 ꢁC to rt overnight. (f) (1) Pd/C (10%, 10 wt %), trioctylamine (0.6 equiv), CH3OH, H2, 24 h. (2) UMP-morpholidate
0
ꢁ
4-morpholine-N-N -dicyclohexylcarboxamide salt (1.5 equiv), 1-H-tetrazole (4 equiv), pyridine, 4 A MS, 3 days. (g) CH3OH, H2O, Et3N (4:2:1), 24 h.

A.E. Dulcey et al. / Tetrahedron 67 (2011) 2013e2017
2015
OAc
OAc
OAc
OAc
OAc
OH
OAc
1. [O]
2. Deprotection
crude mixture was purified by flash column chromatography on silica
gel eluted with a gradient of 3:1/2:1 Hex/EtOAc to afford the un-
wanted 2-iodomannose isomer (lower Rf) as the major product, and
the desired 2-iodoglucose derivative 2-Glc (higher Rf) as the minor
O
O
OH
OAc
+
AcO
AcO
AcO
O
O
3a-Gal
3-Gal
3b-Gal
product (634 mg, 25% yield) as a clear syrup with an
a
:
b
anomeric
ratio of 1:10.
b
anomer: 1H NMR (400 MHz, CDCl3)
d
5.85 (d, J¼9.6 Hz,
OAc
OAc
1. Phosphoramidite
2. [O]
O
1H), 5.32 (t, J¼10.0 Hz, 1H), 4.99 (t, J¼10.0 Hz, 1H), 4.30 (dd, J¼12.4,
4.4 Hz, 1H), 4.07 (dd, J¼12.4, 2.0 Hz, 1H), 3.97 (t, J¼11.2 Hz, 1H), 3.86
(ddd, J¼10.0, 4.4, 2.0 Hz,1H), 2.15 (s, 3H), 2.08(s, 3H), 2.06 (s, 3H), 2.00
AcO
O
O
P
OBn
O
OBn
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 170.5, 169.7, 169.4, 168.4, 93.8,
6-Gal
75.1, 72.9, 68.4, 61.4, 25.6, 20.6, 20.5; MS (m/z)¼459.7 (Mþ1)þ; HRMS
Scheme 2. Problematic step and unwanted side-product generation via Aldol
(ESI) calcd for C14H19IO9Naþ [MþNa]þ 480.9966, found 480.9966.
condensation.
glucose analog 8-Glc was synthesized in eight steps and 4.8%
overall yield, and the UDP-2-(2-ketopropyl)galactose analog 8-Gal
was obtained in the same number of steps with and overall yield of
5.3%. With the latter previously described compound a 7.5-fold
yield improvement was obtained over the first generation syn-
thesis,2 which also involved eight steps but resulted in an overall
yield of only 0.7%.
4.1.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-iodo-
D
-galactopyranose (2-
Gal). Tri-O-acetyl- -galactal (2.30 g, 8.45 mmol), NIS (2.28 g,
D
10.1 mmol), AcOH (1 mL, 16.9 mmol) and, CH2Cl2 (25 mL) to obtain
2-Gal (1.34 g, 34% yield) as syrup, minor product with higher Rf.
b
anomer: 1H NMR (400 MHz, CDCl3)
d
5.88 (d, J¼9.2 Hz, 1H), 5.32
(m, 1H), 5.22 (d, J¼4 Hz, 1H), 5.12 (dd, J¼12.0, 3.6 Hz, 1H), 4.33 (t,
J¼6.8 Hz, 1H), 4.11 (m, 1H), 4.04 (m, 1H), 2.15 (s, 3H), 2.11 (s, 3H),
2.04 (s, 3H), 2.01 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 170.2, 169.8,
4. Experimental section
4.1. General
169.5, 168.4, 93.3, 73.0, 72.3, 68.8, 61.1, 24.7, 20.5, 19.5; MS (m/z)¼
459.1 (Mþ1)þ; HRMS (ESI) calcd for C14H19IO9Naþ [MþNa]þ
480.9966, found 480.9973..
All commercially available organic precursors and dry solvents
were obtained from SigmaeAldrich, and used as received unless
otherwise noted. Reactions were magnetically stirred under an ar-
gon atmosphere and monitored by thin layer chromatography (TLC)
with 0.25 mm SigmaeAldrich pre-coated aluminum-backed silica
gel plates with fluorescent indicator. TLC visualization was achieved
using 254 nm or 360 nm UV lamp detection and/or staining with
cerium molybdate (Hannesian’s stain), phosphomolybdic acid
(PMA), or potassium permanganate. Flash column chromatography
was performed on an AnaLogix IntelliFlash 280 system, using Bio-
tageÒ SNAP Cartridges and SNAP Samplet Cartridges with KP-Silica
4.1.3. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(methallyl)-D-glucopyranose
(3-Glc). A solution of 2-Glc (1.30 g, 2.84 mmol) and methallyl-
tributylstannane9 (1.96 g, 5.67 mmol) in benzene (15 mL) was
thoroughly degassed by bubbling argon through it for 20 min. 2,20-
Azobis(2-methylpropionitrile) (AIBN) (70.0 mg, 0.43 mmol) was
then added in one portion and degassing continued for another
5 min. The resulting reaction mixture was refluxed for 3 h, after
which TLC (2:1 Hex/EtOAc) showed completion. The reaction
mixture was allowed to cool to room temperature and concen-
trated under reduced pressure. The residue was taken up in ace-
tonitrile and washed with hexanes (three times). The acetonitrile
layer was concentrated and the residue purified by flash column
chromatography on silica gel using 2:1 Hex/EtOAc to afford 3-Glc as
60
Series instrument equipped with multi-wavelength detectors using
a Zorbax Stable Bond C-18 column (4.6ꢂ50 mm, 3.5 m) with a flow
mm. Analytical HPLC analyses were performed on an Agilent 1200
m
a clear oil (768 mg, 70% yield). b
anomer: 1H NMR (400 MHz, CDCl3)
rateof 0.5 mL/min or 1.0 mL/min. Solvent Awas 0.05% trifluoroacetic
acid (TFA) in water (H2O), solvent B was 0.05% TFA in acetonitrile
(ACN), and a linear gradient of 5e95% B over 10 min was used. ESI or
APCI mass spectrometry (MS) was performed on an LC/MSD TrapXCl
Agilent Technologies instrument or on a 6130 Quadrupole LC/MS
Agilent Technologies instrument equipped with a diode array de-
tector. High resolution mass spectrometry (HRMS) was performed
by The Scripps Research Institute Center for Metabolomics and Mass
Spectrometry (La Jolla, CA). 1H, 13C and 31P NMR spectra were
recorded on a Varian spectrometer operating at 400 MHz, 100 MHz
and 162 MHz, respectively. Chemical shifts are reported relative to
d
5.54 (d, J¼8.8 Hz, 1H), 5.01 (t, J¼6.8 Hz, 1H), 4.67 (s, 1H), 4.62 (s,
1H), 4.31 (dd, J¼12.4, 4.4 Hz, 1H), 4.06 (dd, J¼12.4, 2.4 Hz, 1H),
3.79e3.74 (m, 1H), 2.10e2.07 (m, 10H), 2.00 (s, 3H), 1.98 (s, 3H), 1.73
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 170.6, 170.2, 169.8, 168.9, 142.3,
112.2, 94.3, 72.3, 69.1, 61.9, 42.1, 37.1, 21.6, 20.8, 20.7, 20.6; MS (m/
z)¼387.9 (Mþ1)þ; HRMS (ESI) calcd for C18H26O9Naþ [MþNa]þ
409.1469, found 409.1467.
4.1.4. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(methallyl)-D-galactopyr-
anose (3-Gal). 2-Gal (2.20 g, 4.80 mmol), methallyltributyl-
stannane8 (4.14 g, 12.0 mmol), AIBN (118 mg, 0.72 mmol), and
benzene (30 mL) afforded 3-Gal (1.26 g, 68% yield) as a clear oil.
either chloroform (
2.05) ordeuteriumoxide (
dimethyl sulfoxide ( 39.5) or acetone (
d
7.26), dimethyl sulfoxide (
4.79)for 1HNMRand chloroform(
29.8) for 13C NMR.
d
2.50), acetone (
d
d
d
77.0),
b
anomer: 1H NMR (400 MHz, CDCl3)
d
5.54 (d, J¼9.2 Hz, 1H),
d
d
5.36e5.33 (m, 1H), 5.26 (d, J¼2.4 Hz, 1H), 4.82 (dd, J¼11.6, 3.2 Hz,
1H), 4.66 (s, 1H), 4.59 (s, 1H), 4.16e4.03 (m, 3H), 3.94 (t, J¼6.8 Hz,
1H), 2.12e2.09 (m, 4H), 2.05 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.72 (s,
4.1.1. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-iodo-
N-Iodosuccinimide (NIS) (2.04 g, 9.07 mmol) was added at room
temperature to solution of tri-O-acetyl- -galactal (1.51 g,
5.55 mmol) and acetic acid (AcOH) (635 L, 11.1 mmol) in dry
D-glucopyranose (2-Glc).
3H); 13C NMR (100 MHz, CDCl3)
d 170.2, 169.9, 168.8, 142.6, 113.6,
a
D
113.0, 112.1, 93.7, 71.4, 68.9, 67.0, 66.8, 37.9, 36.8, 35.7, 21.8, 20.9,
20.7, 20.3, 19.7; MS (m/z)¼387.5 (Mþ1)þ; HRMS (ESI) calcd for
C18H26O9Naþ [MþNa]þ 409.1469, found 409.1475.
m
dichloromethane (CH2Cl2) (20 mL) in an aluminum foil-wrapped
flask. The resulting reaction mixturewasallowedto stirinthe dark for
45 min, after which TLC [2:1 hexane (Hex)/ethyl acetate (EtOAc)]
showed completion. The reaction was quenched by the addition of
a saturated aqueous solution of sodium thiosulfate (Na2S2O3) and
allowed to stir for 15 min. The reaction mixture was diluted with
CH2Cl2 and washed with Na2S2O3 (one time), H2O (one time) and
brine, dried over magnesium sulfate (MgSO4), and concentrated. The
4.1.5. 3,4,6-Tri-O-acetyl-2-deoxy-2-(methallyl)- -glucopyranose (4-
D
Glc). Hydrazine acetate (102 mg, 1.11 mmol) was added in one
portion to a solution of 3-Glc (357 mg, 0.92 mmol) in N,N-dime-
thylformamide (DMF) (5 mL). The reaction was allowed to stir at
room temperature for 2 h. TLC (2:1 Hex/EtOAc) showed comple-
tion. The reaction mixture was partitioned between H2O and EtOAc,

2016
A.E. Dulcey et al. / Tetrahedron 67 (2011) 2013e2017
and the organic layer washed with H2O (three times), followed by
brine, dried over MgSO4, and concentrated under reduced pressure.
Product was purified by flash column chromatography on silica gel
with 2:1 Hex/EtOAc to afford the lactol 4-Glc as a syrup (265 mg,
607.3 (Mþ1)þ; HRMS calcd for C29H35O12Pþ [MþH]þ 607.1939,
found 607.1935.
4.1.9. Uridine 50-diphospho-2-acetonyl-2-deoxy-
a-D-glucopyranose
83% yield).
a
anomer: 1H NMR (400 MHz, CDCl3)
d
5.27 (t,
diammonium slat (8-Glc). Pd/C (10%, 30 mg) was added to a solu-
tion of 6-Glc (290 mg, 0.48 mmol) and trioctylamine (101 mg,
0.29 mmol) in dry CH3OH (4 mL). The resulting reaction mixture
was subjected to an H2 atmosphere via a balloon and allowed to stir
for 20 h. TLC (1:1 Hex/EtOAc) showed complete disappearance of 6-
Glc starting material. The reaction mixture was filtered through
CeliteÔ and concentrated to dryness. The residue was taken up in
dry pyridine (3 mL) and UMP-morpholidate 4-morpholine-N,N0-
dicyclohexylcarboxamide salt (492 mg, 0.72 mmol) was added
J¼10.0 Hz, 1H), 5.15 (d, J¼3.2 Hz, 1H), 4.96 (t, J¼9.6 Hz, 1H),
4.78e4.68 (m, 3H), 4.26e4.21 (m, 1H), 4.17e4.12 (m, 1H), 4.09e4.02
(m, 1H), 3.33 (d, J¼3.6 Hz, 1H), 2.07e2.04 (m, 5H), 2.01 (s, 3H), 2.01
(s, 3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.69 (s, 3H); 13C NMR (100 MHz,
CDCl3)
d 170.9, 170.6, 170.1, 142.4, 114.0, 112.5, 93.6, 72.7, 71.3, 70.5,
69.3, 68.5, 67.7, 65.7, 62.8, 42.2, 40.0, 35.9, 23.1, 21.6, 20.6, 19.7; MS
(m/z)¼345.8 (Mþ1)þ; HRMS (ESI) calcd for C16H24O8Naþ [MþNa]þ
367.1363, found 367.1368.
ꢁ
along with activated 4 A molecular sieves (w300 mg), followed by
4.1.6. 3,4,6-Tri-O-acetyl-2-deoxy-2-(methallyl)-
D
-galactopyranose
the addition of 1H-tetrazole (134 mg, 1.91 mmol) in pyridine
(2 mL). The resulting reaction mixture was allowed to stir at room
temperature for 3 days and then filtered through CeliteÔ. The fil-
trate was concentrated under reduced pressure and the residue
taken up in CH3OH (4 mL). H2O (2 mL) was added, followed by Et3N
(1 mL). The resulting reaction mixture was allowed to stir at room
temperature for 24 h then diluted with H2O and extracted with
CH2Cl2 (two times). The aqueous layer was lyophilized and the
crude mixture purified by HPLC using a preparative Agilent Zorbax
(4-Gal). Hydrazine acetate (145 mg, 1.56 mmol), 3-Gal (550 mg,
1.42 mmol), and DMF (5 mL) to obtain 4-Gal (343 mg, 70% yield).
a
anomer: 1H NMR (400 MHz, CDCl3)
d
5.31 (d, J¼2.8 Hz, 1H), 5.23
(t, J¼3.2 Hz, 1H), 5.14 (dd, J¼11.6, 3.2 Hz, 1H), 4.77 (d, J¼10.0 Hz,
2H), 4.41 (t, J¼6.4 Hz, 1H), 4.13e4.11 (m, 1H), 4.07 (t, J¼5.6 Hz, 1H),
2.98 (d, J¼3.6 Hz, 1H), 2.14e2.10 (m, 5H), 2.03 (s, 3H), 1.97 (s, 3H),
1.75e1.69 (m, 3H); 13C NMR (100 MHz, CDCl3)
d 170.6, 170.4, 142.2,
112.5, 93.2, 69.5, 67.1, 66.5, 62.4, 36.4, 35.3, 22.6, 22.3, 20.7 (ꢂ2); MS
(m/z)¼345.6 (Mþ1)þ; HRMS (ESI) calcd for C16H24O8Naþ [MþNa]þ
367.1363, found 367.1369.
XDB C-18 column, 21.4ꢂ250 mm, 5
mm, eluted with 100 mM am-
monium bicarbonate buffer (NH4HCO3, pH¼7.9) at a rate of 20 mL/
min. The desired product eluted at a retention time of 4.1e6.2 min
to afford 8-Glc (150 mg, 49% yield) as a white powder after ly-
4.1.7. Dibenzyl (2-acetonyl-2-deoxy-3,4,5-tri-O-acetyl-a-D-glucopyr-
anosyl) phosphate (6-Glc). A solution of lactol 4-Glc (250 mg,
0.73 mmol) and 1H-tetrazole (200 mg, 2.90 mmol) in dry CH2Cl2
(5 mL) was cooled to ꢀ30 ꢁC and treated with dibenzyl N,N-diiso-
propylphosphoramidite (627 mg, 1.81 mmol). The resulting re-
action mixture was allowed to slowly warm from ꢀ30 ꢁC to room
temperature for 3 h. TLC (2:1 Hex/EtOAc) showed complete dis-
appearance of lactol 4-Glc. The reaction mixture was diluted with
Et2O and washed with cold brine (two times), dried over MgSO4,
and concentrated under reduced pressure. The residue was then
taken up in dry CH2Cl2 (6 mL) and cooled to ꢀ78 ꢁC. A stream of
ozone was bubbled through the reaction mixture until a light blue
color became evident (w30e40 min). Argon was then bubbled
through the reaction mixture until the blue color disappeared and
Me2S (1 mL) added. The resulting reaction mixture was allowed to
stir overnight as the temperature rose to ambient. The solvent was
removed under reduced pressure and the residue purified by flash
column chromatography: silica gel,1:1 Hex/EtOAc to afford 6-Glc as
ophilization. 1H NMR (400 MHz, D2O)
d
7.93 (d, J¼8.0 Hz, 1H),
6.00e5.95 (m, 2H), 5.57 (dd, J¼7.2, 3.2 Hz, 1H), 4.40e4.34 (m, 2H),
4.28e4.26 (m, 1H), 4.24e4.16 (m, 2H), 3.91e3.78 (m, 3H), 3.63 (t,
J¼9.2 Hz, 1H), 3.46 (t, J¼9.2 Hz, 1H), 2.86 (dd, J¼18.0, 5.2 Hz, 1H),
2.79e2.72 (m, 2H), 2.62 (s, 3H); 13C NMR (100 MHz, D2O)
d 215.5,
167.2, 151.0, 141.9, 103.5, 96.2, 88.5, 84.6, 74.3, 72.1, 69.6, 68.2, 65.2,
63.9, 63.2, 44.5, 40.6, 30.1; 31P NMR (162 MHz, D2O)
d
ꢀ11.24 (d,
J¼18.0 Hz), ꢀ12.33 (d, J¼19.2 Hz); MS (m/z)¼605.0 (Mþ1)þ; HRMS
(ESI) calcd for C18H26N2O17Pþ2 [MꢀH]ꢀ 605.0785, found 605.0801.
4.1.10. Uridine 50-diphospho-2-acetonyl-2-deoxy-
ose diammonium salt (8-Gal). 6-Gal (60 mg, 99.0
a
-
m
D
-galactopyran-
mol), trioctyl-
amine (21 mg, 59.4 mmol), 10% Pd/C (8 mg), and CH3OH (3 mL).
Then UMP-morpholidate 4-morpholine-N,N0-dicyclohexylcarbox-
amide salt (102 mg, 0.15 mmol), 1H-tetrazole (28 mg, 0.40 mmol),
ꢁ
pyridine (3 mL), activated 4 A molecular sieves (w300 mg). Then
CH3OH (2 mL), H2O (1 mL), Et3N (0.5 mL). Preparative HPLC con-
a syrup (298 mg, 68% yield). 1H NMR (400 MHz, CDCl3)
d
7.37e7.32
ditions same as for 8-Glc, afforded 8-Gal as white powder (34 mg,
(m, 10H), 5.81 (dd, J¼6.4, 3.2 Hz, 1H), 5.37 (t, J¼3.2 Hz, 1H), 5.23 (t,
J¼10.2 Hz, 1H), 5.09e4.97 (m, 4H), 4.21 (t, J¼4.4 Hz, 1H), 4.14e4.06
(m, 2H), 4.04e3.99 (m, 1H), 3.88 (dd, J¼12.4, 2.0 Hz, 1H), 3.31 (d,
J¼2.4 Hz, 1H), 2.13 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.92 (s, 3H); 13C
53% yield) after lyophilization. 1H NMR (400 MHz, D2O)
d 7.95 (d,
J¼8.1 Hz, 1H), 5.97e5.95 (m, 2H), 5.57 (dd, J¼7.5, 3.4 Hz, 1H),
4.35e4.30 (m, 2H), 4.25e4.23 (m, 1H), 4.21e4.16 (m, 2H), 4.11 (t,
J¼5.3 Hz, 1H), 3.91e3.88 (m, 1H), 3.78e3.70 (m, 3H), 2.80e2.75 (m,
NMR (100 MHz, CDCl3)
d
205.5, 170.5,170.4,169.5, 135.3,128.7 (ꢂ2),
1H), 2.56 (m, 1H), 2.60 (s, 3H); 13C NMR (100 MHz, D2O)
d 214.5,
128.0, 97.1, 70.5, 69.7 (ꢂ2), 69.6, 68.6, 61.6, 39.7, 29.7, 20.6, 20.5; 31P
167.9, 152.0, 142.1, 104.1, 96.9, 88.4, 84.1, 74.3, 72.1, 69.8, 68.3, 65.2,
NMR (162 MHz, CDCl3)
d
ꢀ2.31; MS (m/z)¼607.9 (Mþ1)þ; HRMS
64.0, 63.3, 44.6, 40.1, 30.5; 31P NMR (162 MHz, D2O)
d
ꢀ10.24 (d,
(ESI) calcd for C29H35O12Pþ [MþH]þ 607.1939, found 607.1930.
J¼19.0 Hz), ꢀ12.43 (d, J¼19.9 Hz); MS (m/z)¼605.4 (Mþ1)þ; HRMS
(ESI) calcd for C18H26N2O17Pþ2 [MꢀH]ꢀ 605.0785, found 605.0798.
4.1.8. Dibenzyl (2-acetonyl-2-deoxy-3,4,5-tri-O-acetyl-
yranosyl) phosphate (6-Gal). 4-Gal (60 mg,
a
-D-galactop-
0.17 mmol),
Acknowledgements
1H-tetrazole (49 mg, 0.70 mmol), dibenzyl N,N-diisopropylphos-
phoramidite (150 mg, 0.44 mmol), ozone, and Me2S (1 mL) to afford
6-Gal as a syrup (63 mg, 60% yield). 1H NMR (400 MHz, CDCl3)
This work was supported by the NIH Roadmap for Medical Re-
search Initiative through its establishment of the Imaging Probe
Development Center, administered by the National Heart, Lung,
and Blood Institute.
d
7.35e7.30 (m, 10H), 5.86 (dd, J¼6.0, 2.8 Hz, 1H), 5.29 (s, 1H),
5.09e5.00 (m, 4H), 4.92 (dd, J¼12.4, 3.2 Hz, 1H), 4.25 (t, J¼6.4 Hz,
1H), 4.08e4.03 (m, 2H), 3.95e3.91 (m, 1H), 2.36e2.34 (m, 2H), 2.10
(s, 3H), 1.97 (s, 3H), 1.92 (s, 3H), 1.88 (s, 3H); 13C NMR (100 MHz,
Supplementary data
CDCl3)
92.9, 69.6, 69.5 (ꢂ2), 68.9, 66.7, 66.1, 65.9, 61.7, 40.8, 39.7, 34.4, 30.3,
29.8, 20.6, 20.4; 31P NMR (162 MHz, CDCl3)
ꢀ2.24; MS (m/z)¼
d
205.9,170.2 (ꢂ2),170.0,135.3,128.7 (ꢂ2),128.6,128.0, 97.8,
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2011.01.081. These data include
d

A.E. Dulcey et al. / Tetrahedron 67 (2011) 2013e2017
2017
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MOL files and InChIKeys of the most important compounds de-
scribed in this article.
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