A novel galactosyltransferase inhibitor with dlamino sugar as a pyrophosphate mimic

Article abstract of DOI:10.1002/ejoc.200801285

Conjugates of 2,4-diamino sugars and uridine have been synthesized as galactosyltransferase inhibitors. The relationship between inhibitory activity and the chelalion abilility of the hinge-like diamino sugar towards a metal ion was studied by NMR spectro

Full text of DOI:10.1002/ejoc.200801285

FULL PAPER
DOI: 10.1002/ejoc.200801285
A Novel Galactosyltransferase Inhibitor with Diamino Sugar as a
Pyrophosphate Mimic
Nobuyuki Mitsuhashi[a] and Hideya Yuasa*[a]
Keywords: Bioorganic chemistry / Inhibitors / Glycoconjugates / Enzymes / Chelation
Conjugates of 2,4-diamino sugars and uridine have been
ited a moderate inhibition that ranks between those of UDP
synthesized as galactosyltransferase inhibitors. The relation- and UMP probably through the chelation of the hinge-like
ship between inhibitory activity and the chelation abilility of
the hinge-like diamino sugar towards a metal ion was
studied by NMR spectroscopy. One of the conjugates exhib-
diamino sugar to MnII.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
The biosyntheses of native oligosaccharides are catalyzed
by glycosyltransferases (GTs),[1] which transfer a monosac-
charide from a sugar nucleotide to the non-reducing end of
a precursory oligosaccharide (Figure 1). Because the bio-
syntheses of cell walls or crustaceous shell polysaccharides
are vital for bacteria and insects, the inhibitors of GTs are
potential antibiotics or insecticides.[2] These inhibitors can
also be used in the functional studies of the oligosaccha-
rides of glycoproteins or glycolipids. Thus, a number of po-
tential GT inhibitors have been designed and synthesized[3]
that in most cases comprise a pyrophosphate derivative and
a nucleoside as the essential components, for example, com-
pound 1 (Figure 2).[4] The pyrophosphate group seems in-
dispensable for inhibitory activity because it acts as a strong
chelator to the MnII ion bound within GTs. However, the
pyrophosphate group is labile[5] and thus there have been
efforts to create stable pyrophosphate mimics. In the sim-
plest case, an alkyl chain was substituted (compound 2),
but this exhibited no inhibitory activity.[6] The use of either
malonate or tartrate groups as pyrophosphate mimics has
also been unsuccessful.[7] Interestingly, glucose can substi-
tute for the pyrophosphate moiety of uridine diphosphate
galactose (UDPGal) (compound 3), leaving the binding
ability of the enzyme galactosyltransferase (GalT) almost
the same as the native substrate.[7] However, other efforts to
produce a GalT inhibitor by using a sugar to mimic pyro-
phosphate have failed.[8]
Figure 1. A proposed mechanism for the galactosyl transfer reac-
tion catalyzed by galactosyltransferase (GalT).
We have synthesized 2,4-diaminopentopyranosides (4) as
hinge molecules that change conformation from 4C1 to 1C4,
like a hinge, upon the chelation of metal ions (Table 1).[9]
We envisaged that these compounds would serve as pyro-
phosphate mimics because they can chelate MnII through
the hinge motion[10] and the pyranose structure would be
accommodated into the pyrophosphate cleft of GTs, as
noted with compound 3. The hinge-like motion potentially
endows the inhibitors with induced-fit abilities. In addition,
two different functional groups can be attached to the hinge
sugar in a step-by-step manner through a glycosidation re-
action at C1 and alkylation at 3-OH. These attractive prop-
erties of the hinge molecule prompted us to study the inhi-
[a] Department of Life Sciences, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology,
J2-10, 4259 Nagatsutacho, Midoriku, Yokohama 226-8501, Ja-
pan
Fax: +81-45-924-5850
E-mail: hyuasa@bio.titech.ac.jp
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
1598
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1598–1605

A Novel Galactosyltransferase Inhibitor
shown below. The methylene bridge was incorporated first
as the methylthiomethyl (MTM) ether at 3-OH of the pen-
topyranosides 15 and 17 to give MTM ethers 19 and 20.
The coupling reactions of the MTM ethers 19 and 20 with
2Ј,3Ј-O-isopropylidene-U (21) gave the conjugates 22 and
23. Deprotection of 22 and 23 gave compounds 24 and 25,
and then reduction of the azide functions afforded the tar-
get compounds 7 and 8.
Figure 2. Selected examples of synthetic galactosyltransferase
(GalT) inhibitors.
bition of GalT activity by four hinge sugar–uridine (U)
conjugates 5–8, characterized by single-component-in-
stalled hinges for a starting structure and variations of dif-
ferent linkages and stereochemistries at C1 or C3 of the
hinge molecules (Table 1). We herein report the synthesis of
5–8 and the relationship between the inhibition activities
and chelation abilities of these compounds.
Table 1. The structures of reported (4) and targeted (5–8) 2,4-di-
aminopentopyranosides that change conformation on addition of
a metal ion (MII).
Scheme 1. Reagents and conditions: a) 3-N-2Ј,3Ј-Tri-O-benzoyl-U
(9), TMSOTf/CH2Cl2, –40 °C; b) 28% aq. NH3; c) PPh3, MeOH/
THF/H2O (7.5:6:1).
C1[a]
C3[a]
A
B
4
5
6
7
8
EǞA
AǞE
EǞA
EǞA
EǞA
EǞA
EǞA
EǞA
EǞA
AǞE
H or sugars
H
Me or sugars
U
U
Me
Me
H
–CH2-U
–CH2-U
[a] The orientational change of a substituent at C1 or C3 ac-
companied by the 4C1-to-1C4 flip. EǞA represents the change from
an equatorial to an axial orientation.
Results and Discussion
The C1-linked conjugates 5 and 6 were synthesized by
glycosylation at 5-OH of 3-N-2Ј,3Ј-tri-O-benzoyl-U[11] (9;
Scheme 1). The 1-O-trichloroacetimidate derivative 10[9]
was used as the glycosyl donor to afford α- and β-glycosides
11 and 12. Deacylation of 11 and 12 gave compounds 13
and 14, and then reduction of the azide functionality gave
the target compounds 5 and 6. Methyl 2,4-diazido-β-ribo-
pyranoside (17) was synthesized from methyl 2,4-diazido-β-
Scheme 2. Reagents and conditions: a) Tf2O, 2,6-di-tert-butyl-4-
methylpyridine/CH2Cl2; b) CsOAc/DMF; c) NaOMe/MeOH; d)
PPh3, MeOH/THF/H2O (7.5:6:1); e) CH3SCH2Cl, NaH, Bu4NI/
DMF; f) 2Ј,3Ј-O-isopropylidene-U (21), MeOTf/CH2Cl2; g) 80%
AcOH, 70 °C; h) PPh3/28% aq. NH3/pyridine.
We studied the conformational changes that compounds
xylopyranoside (15) by substitution at C3 to afford acetate 5, 6, 18, 7 and 8 underwent upon addition of a metal ion
1
16, followed by deacetylation at this site (Scheme 2). The by H NMR spectroscopy. Unfortunately, we were unable
azide functions of 17 were reduced to give methyl 2,4-di- to use MnII because it caused precipitation and its para-
amino-β-ribopyranoside (18), which was used to test the magnetism caused fatal signal broadening. We used ZnII
chelation ability of a ribo derivative towards metal ions, as instead because the compounds of our study are expected
Eur. J. Org. Chem. 2009, 1598–1605
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1599

N. Mitsuhashi, H. Yuasa
FULL PAPER
to show chelation abilities towards ZnII that exceed but par- signal-broadening for 7 at 30 °C, which indicates that the
allel those towards MnII, as demonstrated with a hinge- pyranoside assumes a slow conformational exchange be-
4
1
sugar-based metal-ion sensor.[10]
tween the C1 and C4 structures (Figure 4, C). Upon ele-
The effect of the stereochemistry at C1 on the chelation vating the temperature to 70 °C, the signals became sharper
ability was studied with α- and β-xylo-1-U 5 and 6. These and the conformational ratio 4C1/1C4 changed to 5:95. This
4
1
pyranosides stayed almost perfectly in the C1 conforma- high C4 population (95%), in comparison with values of
tions (5: 100%; 6: 96%) in solution (Figure 3, A and B).[12] 33 and 37% for the corresponding 3-O-unsubstituted and
Both anomers underwent slow conformational exchange 3-O-galactosyl derivatives, respectively, was surprising.[9]
upon the addition of 2.0 equiv. of ZnII at 30 °C, as indicated On the other hand, ZnII addition to 8 also caused drastic
by broadened signals. Only the spectrum of β-xylo-1-U 6 changes in the 1H NMR spectrum but the signals remained
exhibited sharp signals at 70 °C, with a 4C1/1C4 ratio of unbroadened even at 30 °C and the J values indicated 100%
61:39 (Figure 3, D), whereas signal-broadening was ob- 1C4 population (Figure 4, D). In short, the most significant
served with α-xylo-1-U 5 (Figure 3, C). These results sug- conformational difference between 7 and 8 in the presence
gest that the conformational exchange of α-xylo-1-U 5 is of ZnII was observed at 30 °C, which suggests that ribo-3-
considerably slower than that of β-xylo-1-U 6, which indi- U 8 has kinetically better chelating ability than xylo-3-U 7.
cates that β-xylo-1-U 6 is much more flexible and has a
After establishing the relative chelating abilities of the
better chelating ability than α-xylo-1-U 5.
two series of diamino sugar-U conjugates 5–8, we examined
4
Compound 18 showed a perfect interchange from C1 to the inhibitory activities of these compounds (1 m) against
1C4 upon addition of 2.0 equiv. of ZnII, whereas in previous GalT in comparison with UDP and uridine monophos-
work with the C3 epimer, methyl 2,4-diamino-β-xylopyr- phate (UMP), known inhibitors of GalT with reported Ki
anoside, partial conformational exchange was observed values of 32 and 450 µ, respectively.[13] Umbelliferyl N-
4
with C1/1C4 of around 67:33. These results indicate that acetyl-β--glucosamine (0.1 m) and UDPGal (0.1 m)
the ribopyranoside 18 has greater conformational flexibility were used as the glycosyl acceptor and donor. The β-xylo-
and thus superior chelation ability than the xylopyranoside. 1-U conjugate 6 exhibited a moderate inhibition (45%),
1
The extraordinary stability of the C4 conformation of 18 which ranks between those of UDP (85%) and UMP
is likely due to the anchoring effect of the equatorial 3-OH. (22%), whereas the corresponding α-glycoside 5 showed less
Similar differences in chelation abilities were observed inhibition (18%) than UMP. On the other hand, the ribo-
between xylo-3-U 7 and ribo-3-U 8, which adopted favour- 3-U conjugate 8 barely showed inhibition (10%), whereas
able 4C1 conformations with 4C1/1C4 ratios of around 90:10 the corresponding xylo derivative 7 exhibited no inhibition.
and 66:34, respectively, in the absence of metal ions (Fig- The trend in the inhibition activities of these compounds,
ure 4, A and B). The addition of 2.0 equiv. of ZnII caused that is, β-xylo-1-U 6 Ͼ α-xylo-1-U 5 and ribo-3-U 8 Ͼ xylo-
1
Figure 3. H NMR spectra for compounds 5 and 6 in the absence of metal ions (A and B) and in the presence of 2.0 equiv. of ZnII at
70 °C (C and D).
1600
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1598–1605

A Novel Galactosyltransferase Inhibitor
1
Figure 4. H NMR spectra for compounds 7 and 8 in the absence of metal ions (A and B) and in the presence of 2.0 equiv. of ZnII at
30 °C (C and D).
3-U 7, parallel that of the conformational flexibility upon results of the conjugate 8 probably originate from the meth-
chelation to ZnII. Therefore, strong chelation towards MnII, ylene spacer, which restricts the chelation to MnII (Figure 5,
associated with the conformational flexibility of the di- B).
amino sugars, might be essential for the inhibitory activity
of these pyrophosphate mimics (Figure 5). Further study is
required to corroborate the relationship between the inhibi-
tion activities and chelation abilities. The poor inhibition
Conclusions
We have developed 2,4-diamino sugars as pyrophosphate
mimics for GalT inhibitors that are stable in their own right
and retain their ability to bind with the enzyme. The inhibi-
tion activities appear to depend on conformational flexibil-
ity and thus the chelation abilities of the hinge-like mole-
cules to bite MnII within the structure of GalT. Chelation
would necessarily induce orientational switching of the un-
joined oxygen atom, O3 for β-xylo-1-U 6 or O1 for ribo-3-
U 8, awaiting attachment of a glycosyl donor or acceptor
unit. When the third component is attached, these com-
pounds may become potential motion-driven inhibitors
with induced-fit inhibition enhancements. Further studies
are necessary to prove the relationship between the chela-
tion ability of the hinge sugar part of the inhibitors and the
inhibition activities.
Experimental Section
General: All solvents and reagents used were reagent grade and, for
cases in which further purification was required, standard pro-
cedures[14] were followed. Solution transfers for which anhydrous
conditions were required were performed under dry argon using
syringes. Thin-layer chromatography (TLC) was performed on pre-
Figure 5. Proposed mechanisms for GalT inhibition for compounds
6 (A) and 8 (B).
Eur. J. Org. Chem. 2009, 1598–1605
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1601

N. Mitsuhashi, H. Yuasa
FULL PAPER
coated silica gel Merck 60-F254 plates (Art 5715) and visualized
by the quenching of fluorescence and/or by charring after spraying
with 1% CeSO4/1.5% (NH4)6Mo7O24·4H2O/10% H2SO4. Column
chromatography was performed on Merck Kieselgel 60 (Art 7734),
Wako gel C-300 or Kanto Silica gel 60N (spherical, neutral) with
the solvent systems specified. Optical rotations were determined
with a Horiba SEPA-200 using a 1 dm length cell. 1H NMR spectra
were recorded at 600 (Bruker AV-600), 400 (Varian Unity-400) or
270 MHz (JEOL EX-270). Internal tetramethylsilane (δ = 0 ppm)
was used as a standard in CDCl3 and solvent peaks were used as
standards in [D6]DMSO (δ = 2.5 ppm) and CD3OD (δ = 3.3 ppm).
Chemical shifts are expressed in ppm referenced to the solvent as
an internal standard. The multiplicity of the signals is abbreviated
as follows: s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, dt = doublet of triplets, ddd = doublet of doublets of dou-
blets, br. = broad signal, m = multiplet. 13C NMR spectra were
recorded at 150 (Bruker AV600) or 67.8 MHz (JEOL JNM-EX-
270) and a solvent peak (δ = 77.0 ppm in CDCl3, δ = 49.0 ppm in
CD3OD, or δ = 39.52 ppm in [D6]DMSO) or acetone (δ =
30.89 ppm in D2O) was used as a standard. High-resolution mass
spectra (HRMS) were recorded with a Mariner Biospectrometry
Workstation ESI-TOF MS.
6 4 . 2 , 6 4 . 0 , 5 9 . 2 , 2 0 . 7 p p m . H R M S ( E S I ) : c a l c d . fo r
C37H32N8NaO12 [M + Na]+ 803.2038; found 803.2063.
5Ј-O-(2,4-Diazido-2,4-dideoxy-α-D-xylopyranosyl)uridine (13): A
28% NH3 (1 mL) solution was added to a stirred solution of 11
(76.0 mg, 0.097 mmol) in MeOH (1 mL) at room temp. After 24 h,
the solution was evaporated and the residue was purified by
chromatography on silica gel (CHCl3/MeOH, 8:1) to give 13
(40.6 mg, 98%) as a foam. Rf = 0.20 (CHCl3/MeOH, 8:1). [α]2D5
104.5 (c = 1.00, MeOH). H NMR (600 MHz, CD3OD): δ = 8.14
=
1
3
3
(d, JH,H = 8.1 Hz, 1 H, 6-H), 5.92 (d, JH,H = 4.6 Hz, 1 H, 1Ј-H),
3
3
5.73 (d, JH,H = 8.1 Hz, 1 H, 5-H), 4.94 (d, JH,H = 3.5 Hz, 1 H,
3
1ЈЈ-H), 4.16–4.15 (m, 1 H, 4Ј-H), 4.13 (t, JH,H = 4.6 Hz, 1 H, 3Ј-
H), 4.11 (t, 3JH,H = 4.6 Hz, 1 H, 2Ј-H), 3.89 (dd, 3JH,H = 2.4, 2JH,H
3
= 11.2 Hz, 1 H, 5aЈ-H), 3.74 (t, JH,H = 9.7 Hz, 1 H, 3ЈЈ-H), 3.73–
3.69 (m, 2 H, 5aЈЈ-H, 5bЈ-H), 3.59–3.55 (m, 2 H, 4ЈЈ-H, 2ЈЈ-H), 3.37
3
(t, JH,H
=
2JH,H = 11.3 Hz, 1 H, 5bЈЈ-H) ppm. 13C NMR
(67.8 MHz, CD3OD): δ = 166.3, 152.4, 142.9, 102.7, 98.9, 90.3,
84.0, 75.9, 73.6, 71.0, 67.6, 65.7, 63.4, 61.2 ppm. HRMS (ESI):
calcd. for C14H19N8O8 [M + H]+ 427.1326; found 427.1335.
5Ј-O-(2,4-Diazido-2,4-dideoxy-β-D-xylopyranosyl)uridine (14): A
28% NH3 (1 mL) solution was added to a stirred solution of 12
(52.4 mg, 0.067 mmol) in MeOH (1 mL) at room temp. After 24 h,
the solution was evaporated and the residue was purified by
chromatography on silica gel (CHCl3/MeOH, 8:1) to give 14
5Ј-O-(3-O-Acetyl-2,4-diazido-2,4-dideoxy-α,β-D-xylopyranosyl)-3-
N-benzoyl-2Ј,3Ј-di-O-benzoyl-uridine (11 and 12): TMSOTf (1.4 µL,
0.008 mmol) was added to a stirred mixture of 3-O-acetyl-2,4-di-
azido-2,4-dideoxy-xylopyranosyl trichloroacetoimidate (10,
32.7 mg, 0.085 mmol), 3-N-benzoyl-2Ј,3Ј-O-dibenzoyluridine (9;
57.1 mg, 0.103 mmol) and 4 Å MS in CH2Cl2 (1 mL) at –60 °C.
After 2 h, the temperature was elevated to –40 °C and the mixture
was further stirred. After 3 h from the start of the reaction, triethyl-
amine (8 µL) was added and the mixture was filtered through Celite
and the solvents evaporated. The residue was purified by
chromatography on silica gel (hexane/EtOAc, 3:1–2:1–1:1) to give
11 (30.4 mg, 46%) and 12 (16.3 mg, 25%) each as a syrup.
(23.2 mg, 81%) as a foam. Rf = 0.20 (CHCl3/MeOH, 8:1). [α]2D5
–24.0 (c = 0.68, MeOH). H NMR (600 MHz, CD3OD): δ = 7.97
=
1
3
3
(d, JH,H = 8.1 Hz, 1 H, 6-H), 5.90 (d, JH,H = 4.6 Hz, 1 H, 1Ј-H),
3
3
5.75 (d, JH,H = 8.1 Hz, 1 H, 5-H), 4.35 (d, JH,H = 8.1 Hz, 1 H,
1ЈЈ-H), 4.23 (t, JH,H = 4.7 Hz, 1 H, 3Ј-H), 4.17–4.14 (m, 3 H, 2Ј-
H, 4Ј-H, 5aЈ-H), 3.98 (dd, JH,H = 5.4, JH,H = 11.7 Hz, 1 H, 5aЈЈ-
3
3
2
3
2
H), 3.81 (dd, JH,H = 2.2, JH,H = 10.6 Hz, 1 H, 5bЈ-H), 3.57–3.53
(m, 1 H, 4ЈЈ-H), 3.43 (t, 3JH,H = 9.5 Hz, 1 H, 3ЈЈ-H), 3.26 (dd, 3JH,H
= 8.1, 3JH,H = 9.5 Hz, 1 H, 2ЈЈ-H), 3.17 (t, 3JH,H = 2JH,H = 11.3 Hz,
1 H, 5bЈЈ-H) ppm. 13C NMR (67.8 MHz, CD3OD): δ = 166.3,
152.4, 142.4, 103.6, 102.6, 90.6, 84.5, 75.9, 75.6, 71.3, 69.8, 68.3,
65.1, 62.9 ppm. HRMS (ESI): calcd. for C14H19N8O8 [M + H]+
427.1326; found 427.1315.
11: Rf = 0.09 (hexane/EtOAc, 2:1). [α]2D5 = –2.7 (c = 1.00, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 8.00–7.32 (m, 16 H, Phϫ3, 6-
3
3
H), 6.48 (d, JH,H = 6.7 Hz, 1 H, 1Ј-H), 5.96 (d, JH,H = 8.2 Hz, 1
3
3
H, 5-H), 5.76 (dd, JH,H = 5.8, JH,H = 3.3 Hz, 1 H, 3Ј-H), 5.63
(dd, JH,H = 6.7, JH,H = 5.8 Hz, 1 H, 2Ј-H), 5.49–5.46 (m, 1 H,
3ЈЈ-H), 5.03 (d, JH,H = 3.5 Hz, 1 H, 1ЈЈ-H), 4.52 (m, 1 H, 4Ј-H),
5Ј-O-(2,4-Diamino-2,4-dideoxy-α-D-xylopyranosyl)uridine (5): Tri-
3
3
phenylphosphane (125 mg, 0.477 mmol) was added to a stirred
solution of 13 (40.6 mg, 0.095 mmol) in MeOH/THF/H2O (7.5:6:1,
1 mL) at room temp. After 9 h, the solution was evaporated and
partitioned between water and CHCl3. The water layer was evapo-
rated and the residue was purified by chromatography on silica gel
(Kanto Co., 60N spherical gel, iPrOH/H2O/28%NH3, 35:3:1) to
give 5 (26.9 mg, 75%) as a white solid. Rf = 0.32 (iPrOH/H2O/
28%NH3, 10:3:1); m.p. 156–158 °C. [α]2D5 = 70.2 (c = 0.56, H2O).
3
3
2
4.15 (dd, JH,H = 2.6, JH,H = 11.0 Hz, 1 H, 5aЈ-H), 3.88–3.84 (m,
2 H, 5bЈ-H, 5aЈЈ-H), 3.73–3.70 (m, 2 H, 4ЈЈ-H, 5bЈЈ-H), 3.64 (dd,
3
3JH,H = 3.5, JH,H = 10.3 Hz, 1 H, 2ЈЈ-H), 2.20 (s, 3 H, Ac) ppm.
13C NMR (150.9 MHz, CDCl3): δ = 169.6, 168.3, 165.4, 165.1,
161.7, 149.5, 140.0, 135.0, 133.8, 133.7, 131.2, 130.5, 129.9, 129.7,
129.7, 129.1, 128.6, 128.5, 128.5, 128.4, 128.3, 103.6, 97.8, 86.5,
81.6, 73.5, 71.4, 71.3, 67.5, 62.1, 60.3, 59.3, 20.7 ppm. HRMS
(ESI): calcd. for C37H32N8O12Na [M + Na]+ 803.2038; found
803.2018.
3
1H NMR (600 MHz, D2O): δ = 7.70 (d, JH,H = 8.0 Hz, 1 H, 6-
3
3
H), 5.85 (d, JH,H = 8.0 Hz, 1 H, 5-H), 5.84 (d, JH,H = 5.0 Hz, 1
3
3
H, 1Ј-H), 4.94 (d, JH,H = 3.5 Hz, 1 H, 1ЈЈ-H), 4.37 (t, JH,H
5.0 Hz, 1 H, 2Ј-H), 4.28 (t, JH,H = 5.0 Hz, 1 H, 3Ј-H), 4.25–4.23
(m, 1 H, 4Ј-H), 3.93 (dd, JH,H = 5.3, JH,H = 11.4 Hz, 1 H, 5aЈ-
=
3
12: Rf = 0.18 (hexane/EtOAc, 2:1). [α]2D5 = –71.2 (c = 1.00, CHCl3).
1H NMR (400 MHz, CDCl3): δ = 8.03–7.27 (m, 16 H, Phϫ3, 6-
3
2
3
2
H), 3.78 (dd, JH,H = 2.4, JH,H = 11.4 Hz, 1 H, 5bЈ-H), 3.71 (dd,
3
3
H), 6.45 (d, JH,H = 6.9 Hz, 1 H, 1Ј-H), 5.94 (d, JH,H = 8.2 Hz, 1
2
3
2
3JH,H = 5.2, JH,H = 11.3 Hz, 1 H, 5aЈЈ-H), 3.50 (t, JH,H = JH,H
H, 5-H), 5.82 (dd, 3JH,H = 5.6, 3JH,H = 2.3 Hz, 1 H, 3Ј-H), 5.71 (dd,
= 11.3 Hz, 1 H, 5bЈЈ-H), 3.42 (t, 3JH,H = 9.9 Hz, 1 H, 3ЈЈ-H), 2.89–
3
3
3JH,H = 6.9, JH,H = 5.6 Hz, 1 H, 2Ј-H), 5.03 (t, JH,H = 10.1 Hz, 1
3
3
2.84 (m, 1 H, 4ЈЈ-H), 2.80 (dd, JH,H = 3.5, JH,H = 9.9 Hz, 1 H,
2ЈЈ-H) ppm. 13C NMR (67.8 MHz, D2O): δ = 168.4, 153.3, 142.5,
103.0, 99.8, 91.1, 82.6, 74.1, 74.0, 70.0, 67.4, 62.7, 55.7, 52.7 ppm.
HRMS (ESI): calcd. for C14H23N4O8 [M + H]+ 375.1517; found
375.1528.
3
H, 3ЈЈ-H), 4.58 (m, 1 H, 4Ј-H), 4.48 (d, JH,H = 8.1 Hz, 1 H, 1ЈЈ-
3
2
H), 4.32 (dd, JH,H = 2.0, JH,H = 10.8 Hz, 1 H, 5aЈ-H), 4.15–4.08
(m, 2 H, 5bЈ-H, 5aЈЈ-H), 3.71–3.65 (m, 1 H, 4ЈЈ-H), 3.50 (dd, 3JH,H
3
3
= 8.1, JH,H = 10.1 Hz, 1 H, 2ЈЈ-H), 3.30 (t, JH,H
= =
2JH,H
11.5 Hz, 1 H, 5bЈЈ-H), 2.22 (s, 3 H, Ac) ppm. 13 C NMR
(150.9 MHz, CDCl3): δ = 169.6, 168.3, 165.4, 165.4, 161.7, 149.4,
139.4, 135.0, 133.8, 133.7, 131.2, 130.4, 129.8, 129.7, 129.0, 128.6,
128.6, 128.4, 128.2, 103.0, 102.4, 86.8, 82.1, 73.9, 72.7, 72.2, 69.4,
5Ј-O-(2,4-Diamino-2,4-dideoxy-β-D-xylopyranosyl)uridine (6): Tri-
phenylphosphane (71 mg, 0.271 mmol) was added to a stirred solu-
tion of 14 (23.2 mg, 0.054 mmol) in MeOH/THF/H2O (7.5:6:1,
1602
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1598–1605

A Novel Galactosyltransferase Inhibitor
1 mL) at room temp. After 9 h, the solution was evaporated and
partitioned between water and CHCl3. The water layer was evapo-
rated and the residue was purified by chromatography on silica gel
(Kanto Co., 60N spherical gel, iPrOH/H2O/28%NH3, 35:3:1) to
give 6 (14.7 mg, 72%) as a white solid. Rf = 0.28 (iPrOH/H2O/
28%NH3, 10:3:1); m.p. 153–155 °C. [α]2D5 = –19.2 (c = 0.172, H2O).
1), 68.0 (C-3), 62.5 (C-2), 60.9 (C-5), 58.0 (C-4), 56.6 (OMe) ppm.
HRMS (ESI): calcd. for C6H10N6O3Na [M + Na]+ 237.0712; found
237.0725.
Methyl 2,4-Diamino-2,4-dideoxy-β-D-ribopyranoside (18): Tri-
phenylphosphane (56.6 mg, 0.216 mmol) was added to a stirred
solution of 17 (22.0 mg, 0.103 mmol) in MeOH/THF/H2O (7.5:6:1,
0.5 mL). After 6 h, the solution was diluted with CHCl3 and ex-
tracted with H2O. The water layer was evaporated and the residue
was purified by chromatography on silica gel (Kanto Co., 60N
spherical gel, iPrOH/H2O/28%NH3, 35:3:1) to give 18 (42.4 mg,
91%) as a white solid. Rf = 0.22 (iPrOH/H2O/28%NH3, 35:3:1);
3
1H NMR (600 MHz, D2O): δ = 7.88 (d, JH,H = 8.2 Hz, 1 H, 6-
3
3
H), 5.89 (d, JH,H = 4.8 Hz, 1 H, 1Ј-H), 5.87 (d, JH,H = 8.2 Hz, 1
3
3
H, 5-H), 4.41 (d, JH,H = 8.1 Hz, 1 H, 1ЈЈ-H), 4.36 (t, JH,H
4.8 Hz, 1 H, 2Ј-H), 4.32 (t, JH,H = 4.8 Hz, 1 H, 3Ј-H), 4.25 (m, 1
H, 4Ј-H), 4.19 (d, JH,H = 11.5 Hz, 1 H, 5aЈ-H), 4.01 (dd, JH,H
5.0, JH,H = 11.5 Hz, 1 H, 5aЈЈ-H), 3.89 (dd, JH,H = 4.1, JH,H
=
3
2
3
=
=
2
3
2
m.p. 64–65 °C. [α]2D8 = –89.0 (c = 0.67, H2O). H NMR (400 MHz,
1
3
2
11.5 Hz, 1 H, 5bЈ-H), 3.32 (t, JH,H = JH,H = 11.4 Hz, 1 H, 5bЈЈ-
3
3
D2O): δ = 4.37 (d, JH,H = 8.4 Hz, 1 H, 1-H), 3.95 (t, JH,H
=
H), 3.28 (t, 3JH,H = 9.6 Hz, 1 H, 3ЈЈ-H), 2.91 (dt, 3JH,H = 5.0, 3JH,H
2.9 Hz, 1 H, 3-H), 3.74 (dd, 3JH,H = 5.2, JH,H = 11.4 Hz, 1 H, 5a-
H), 3.53 (s, 3 H, OMe), 3.50 (t, JH,H = JH,H = 11.0 Hz, 1 H, 5b-
H), 2.96 (ddd, JH,H = 2.7, JH,H = 5.2, JH,H = 11.0 Hz, 1 H, 4-
2
3
= 10.1 Hz, 1 H, 4ЈЈ-H), 2.68 (t, JH,H = 8.9 Hz, 1 H, 2ЈЈ-H) ppm.
3
2
13C NMR (67.8 MHz, D2O): δ = 167.6, 152.8, 142.6, 104.3, 102.7,
90.4, 83.2, 75.9, 74.3, 70.0, 69.2, 66.4, 57.5, 52.6 ppm. HRMS
(ESI): calcd. for C14H23N4O8 [M + H]+ 375.1517; found 375.1518.
3
3
3
H), 2.57 (dd, JH,H = 8.4, JH,H = 3.0 Hz, 1 H, 2-H) ppm. 13C
NMR (67.8 MHz, D2O): δ = 103.5 (C-1), 71.3 (C-3), 65.3 (C-5),
57.8 (OMe), 54.6 (C-2), 50.0 (C-4) ppm. HRMS (ESI): calcd. for
C6H15N2O3 [M + H]+ 163.1083; found 163.1081.
3
3
1
Compound 6 (16 m
M
) + 2.0 Equiv. Zn(OAc)2: H NMR (600 MHz,
3
50 m [D3]AcONa buffer, pH 7.0, 70 °C): δ = 8.26 (d, JH,H
=
8.1 Hz, 1 H, 6-H), 6.34 (d, 3JH,H = 8.1 Hz, 1 H, 5-H), 6.34 (d, 3JH,H
18 (16 m
[D3]AcONa buffer, pH 7.0, 70 °C): δ = 5.34 (s, 1 H, 1-H), 4.65 (t,
M
) + 2.0 Equiv. Zn(OAc)2: 1H NMR (400 MHz, 50 m
3
= 5.1 Hz, 1 H, 1Ј-H), 5.08 (d, JH,H = 5.0 Hz, 1 H, 1ЈЈ-H), 4.75 (t,
3JH,H = 5.5 Hz, 1 H, 3Ј-H), 4.72–4.67 (m, 1 H, 4Ј-H), 4.63 (dd,
3
2
3JH,H = 4.4 Hz, 1 H, 3-H), 4.55 (dd, JH,H = 1.7, JH,H = 13.0 Hz,
3JH,H = 4.0, JH,H = 12.2 Hz, 1 H, 5aЈЈ-H), 4.60 (dd, JH,H = 2.6,
2
3
3
2
2JH,H = 11.7 Hz, 1 H, 5aЈ-H), 4.33 (dd, JH,H = 4.6, JH,H
=
3
2
1 H, 5a-H), 4.25 (dd, JH,H = 2.0, JH,H = 13.0 Hz, 1 H, 5b-H),
3
3
3.95 (m, 4 H, 4-H, OMe), 3.89 (d, JH,H = 4.4 Hz, 1 H, 2-H), 2.43
11.7 Hz, 1 H, 5bЈ-H), 4.05 (br. t, JH,H = 7.0 Hz, 1 H, 3ЈЈ-H), 3.96
(dd, 3JH,H = 8.0, 2JH,H = 12.2 Hz, 1 H, 5bЈЈ-H), 3.58 (br., 1 H, 4ЈЈ-
H), 3.40 (br., 1 H, 2ЈЈ-H), 2.38 (s, 12 H, Ac) ppm.
(s, 12 H, Ac) ppm.
Methyl 2,4-Diazido-2,4-dideoxy-3-O-methylthiomethyl-β-D-xylopyr-
anoside (19): Compound 15 (94.7 mg, 0.442 mmol) and then meth-
ylthiomethyl chloride (55 µL) were added to a stirred mixture of
55% NaH (118.6 mg, 2.72 mmol), prewashed three times with hex-
ane, in THF (5 mL). After 9 h, methanol was added and the mix-
ture was evaporated. The residue was purified by chromatography
on silica gel (hexane/EtOAc, 12:1) to give 19 (85.5 mg, 71%) as a
syrup. Rf = 0.55 (hexane/EtOAc, 4:1). [α]2D5 = –26.2 (c = 0.508,
Methyl 3-O-Acetyl-2,4-diazido-2,4-dideoxy-β-D-ribopyranoside (16):
2,6-Di-tert-butyl-4-methylpyridine (83 mg, 0.402 mmol) and Tf2O
(68 µL, 0.402 mmol) were added to a stirred solution of methyl 2,4-
diazido-2,4-dideoxy-β--ribopyranoside (15; 28.7 mg, 0.134 mmol)
in CH2Cl2 (1 mL) at room temp. After 1 h, the solution was diluted
with CHCl3 and washed with H2O. The organic layer was dried
with MgSO4, filtered and the solvents evaporated. The residue was
dissolved in DMF (1 mL) and CsOAc (38.5 mg, 0.201 mmol) was
added. After stirring for 2 h at room temp., the mixture was evapo-
rated and the residue was purified by chromatography on silica gel
(hexane/EtOAc, 8:1–3:1) to give 16 (27.8 mg, 81%) as crystals. Rf
= 0.43 (hexane/EtOAc, 3:1); m.p. 57–58 °C. [α]2D8 = –81.3 (c = 1.00,
1
CHCl3). H NMR (270 MHz, CDCl3): δ = 4.95 (m, 2 H, SCH2O),
3
3
2
4.13 (d, JH,H = 7.7 Hz, 1 H, 1-H), 4.03 (dd, JH,H = 5.1, JH,H
=
11.9 Hz, 1 H, 5a-H), 3.56 (s, 3 H, OMe), 3.51 (m, 1 H, 4-H), 3.42
(t, 3JH,H = 9.4 Hz, 1 H, 3-H), 3.32 (dd, 3JH,H = 7.7, 3JH,H = 9.4 Hz,
3
2
1 H, 2-H), 3.17 (dd, JH,H = 10.6, JH,H = 11.9 Hz, 1 H, 5b-H),
2.27 (s, 3 H, MeS) ppm. 13C NMR (67.8 MHz, CDCl3): δ = 103.8
(C-1), 77.7 (C-3), 76.6 (SCH2O), 65.8 (C-2), 63.9 (C-5), 60.9 (C-4),
57.2 (OMe), 14.5 (MeS) ppm. HRMS (ESI): calcd. for
C8H14N6NaO3S [M + Na]+ 297.0746; found 297.0744.
3
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 5.49 (t, JH,H = 3.4 Hz,
3
3
1 H, 3-H), 4.64 (d, JH,H = 4.6 Hz, 1 H, 1-H), 3.93 (dd, JH,H
=
2
2.7, JH,H = 11.3 Hz, 1 H, 5a-H), 3.78 (m, 1 H, 4-H), 3.74 (dd,
3JH,H = 6.1, JH,H = 11.3 Hz, 1 H, 5b-H), 3.67 (dd, JH,H = 4.6,
3JH,H = 3.4 Hz, 1 H, 2-H), 3.48 (s, 3 H, OMe), 2.19 (s, 3 H,
Ac) ppm. 13C NMR (67.8 MHz, CDCl3): δ = 169.5 (CH3C=O),
100.1 (C-1), 68.8 (C-3), 61.1 (C-5), 59.7 (C-2), 56.4 (C-4), 56.0
(OMe), 20.5 (CH3 C=O) ppm. HRMS (ESI): calcd. for
C8H12N6O4Na [M + Na]+ 279.0818; found 279.0841.
2
3
Methyl 2,4-Diazido-2,4-dideoxy-3-O-methylthiomethyl-β-D-ribopyr-
anoside (20): Compound 17 (317.3 mg, 1.48 mmol) and then meth-
ylthiomethyl chloride (150 µL) and Bu4NI (206.9 mg, 0.4 equiv.)
were added to a stirred mixture of 55 % NaH (821.1 mg,
18.8 mmol), prewashed three times with hexane, in DMF (10 mL).
After 3.5 h, methanol and then CHCl3 were added and the mixture
was washed with H2O. The organic layer was dried with MgSO4
Methyl 2,4-Diazido-2,4-dideoxy-β-
D-ribopyranoside (17): A 0.1 
solution of NaOMe (0.12 mL) was added to a stirred solution of
16 (30 mg, 0.12 mmol) in methanol (0.5 mL) . After 30 min, the and the solvents evaporated. The residue was purified by
solution was neutralized through Dowex50(H+) and the solvents
evaporated. The residue was purified by chromatography on silica
gel (hexane/EtOAc, 3:1) to give 17 (22.0 mg, 88%) as a white solid.
Rf = 0.29 (hexane/EtOAc, 3:1); m.p. 71–72 °C. [α]2D8 = –69.5 (c =
chromatography on silica gel (hexane/EtOAc, 8:1) to give 20
(331.8 mg, 82%) as crystals. Rf = 0.42 (hexane/EtOAc, 3:1); m.p.
61–63 °C. [α]2D5 = –68.8 (c = 1.00, CHCl3). 1H NMR (400 MHz,
3
CDCl3): δ = 4.85 (s, 2 H, SCH2O), 4.66 (d, JH,H = 3.6 Hz, 1 H,
1
3
3
3
1.00, CHCl3). H NMR (400 MHz, CDCl3): δ = 4.70 (d, JH,H
6.4 Hz, 1 H, 1-H), 4.22 (t, JH,H = 3.1 Hz, 1 H, 3-H), 3.91 (dd,
=
1-H), 4.31 (t, JH,H = 3.6 Hz, 1 H, 3-H), 3.87 (dd, JH,H = 3.5,
3
2JH,H = 11.8 Hz, 1 H, 5a-H), 3.77 (dd, 3JH,H = 5.9, 2JH,H = 11.8 Hz,
1 H, 5b-H), 3.72–3.69 (m, 1 H, 4-H), 3.62 (t, JH,H = 3.6 Hz, 1 H,
2
3
3
3JH,H = 5.0, JH,H = 11.8 Hz, 1 H, 5a-H), 3.87 (dd, JH,H = 8.0,
2JH,H = 11.8 Hz, 1 H, 5b-H), 3.56–3.52 (m, 1 H, 4-H), 3.53 (s, 3 2-H), 3.46 (s, 3 H, OMe), 2.25 (s, 3 H, CH3S) ppm. 13C NMR
H, OMe), 3.43 (dd, JH,H = 6.4, JH,H = 3.1 Hz, 1 H, 2-H), 2.74
3
3
(67.8 MHz, CDCl3): δ = 100.3 (C-1), 75.1 (CH2), 72.2 (C-3), 61.2
(C-5), 60.7 (C-2), 57.4 (C-4), 56.1 (OMe), 14.2 (SMe) ppm. HRMS
(br., 1 H, OH) ppm. 13C NMR (67.8 MHz, CDCl3): δ = 100.3 (C-
Eur. J. Org. Chem. 2009, 1598–1605
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1603

N. Mitsuhashi, H. Yuasa
FULL PAPER
(ESI): calcd. for C8H14N6NaO3S [M + Na]+ 297.0746; found
3
3
(d, JH,H = 8.0 Hz, 1 H, 6-H), 5.77 (d, JH,H = 5.4 Hz, 1 H, 1Ј-H),
3
297.0748.
5.65 (d, JH,H = 8.0 Hz, 1 H, 5-H), 5.46 (br., 1 H, OH), 5.23 (br.,
3
1 H, OH), 4.91 (s, 2 H, OCH2O), 4.35 (d, JH,H = 7.0 Hz, 1 H, 1ЈЈ-
2Ј,3Ј-O-Isopropylidene-5Ј-[(methyl 2,4-diazido-2,4-dideoxy-β-D-xylo-
H), 4.03 (br., 1 H, 2Ј-H), 4.00–3.97 (m, 3 H, 3Ј-H, 4Ј-H, 5aЈЈ-H),
pyranosid-3-O-yl)methyl]uridine (22): MeOTf (62 µL, 0.546 mmol)
was added to a stirred mixture of 19 (101.6 mg, 0.370 mmol), 2Ј,3Ј-
O-isopropylideneuridine (21; 142.3 mg, 0.501 mmol) and 4 Å MS
(0.8 g) in CH2Cl2 (4 mL) at room temp. After 18 h, further MeOTf
(30 µL, 0.264 mmol) was added. After 39 h from the start of the
reaction, triethylamine (80 µL) was added and the mixture was fil-
tered through Celite and the solvents evaporated. The residue was
purified by chromatography on silica gel (hexane/EtOAc, 3:1–1:1)
to give 22 (184.2 mg, 97%) as a white solid. Rf = 0.19 (hexane/
EtOAc, 1:1); m.p. 60–62 °C. [α]2D5 = –14.2 (c = 1.00, CHCl3). 1H
2
3
2
3.84 (d, JH,H = 10.5 Hz, 1 H, 5aЈ-H), 3.75 (dd, JH,H = 2.6, JH,H
= 10.5 Hz, 1 H, 5bЈ-H), 3.71–3.67 (m, 1 H, 4ЈЈ-H), 3.45–3.40 (m, 5
H, OMe, 2ЈЈ-H, 3ЈЈ-H), 3.24 (t, 3JH,H = 2JH,H = 11.1 Hz, 1 H, 5bЈЈ-
H) ppm. 13C NMR (67.8 MHz, [D6]DMSO): δ = 163.2, 150.8,
140.7, 102.5, 102.0, 95.9, 88.0, 82.8, 77.8, 73.1, 70.2, 68.4, 65.1,
62.9, 60.4, 56.5 ppm. HRMS (ESI): calcd. for C16H22N8NaO9 [M
+ Na]+ 493.1408; found 493.1414.
5Ј-[(Methyl 2,4-diazido-2,4-dideoxy-β-D-ribopyranosid-3-O-yl)meth-
yl]uridine (25): A solution of 23 (42.8 mg, 0.084 mmol) in 80% ace-
tic acid (2 mL) was stirred for 11 h at 70 °C. After evaporation of
the solvent, the residue was purified by chromatography on silica
gel (CHCl3/MeOH, 15:1–12:1–10:1) to give 25 (38.7 mg, 98%) as a
3
NMR (400 MHz, CDCl3): δ = 8.67 (br., 1 H, NH), 7.47 (d, JH,H
3
= 8.1 Hz, 1 H, 6-H), 5.84 (d, JH,H = 2.3 Hz, 1 H, 1Ј-H), 5.72 (d,
3JH,H = 8.1 Hz, 1 H, 5-H), 4.99 (d, JH,H = 6.9 Hz, 1 H, OCH2O),
2
white solid. Rf = 0.34 (CHCl3/MeOH, 10:1); m.p. 60–62 °C. [α]2D5
=
4.92 (d, 2JH,H = 6.9 Hz, 1 H, OCH2O), 4.89 (dd, 3JH,H = 6.3, 3JH,H
1
–15.5 (c = 0.452, CHCl3). H NMR (600 MHz, CDCl3): δ = 7.76
3
3
= 3.7 Hz, 1 H, 3Ј-H), 4.85 (dd, JH,H = 2.3, JH,H = 6.3 Hz, 1 H,
3
3
(d, JH,H = 8.0 Hz, 1 H, 6-H), 5.87 (s, 1 H, 1Ј-H), 5.73 (d, JH,H
=
3
2Ј-H), 4.40 (m, 1 H, 4Ј-H), 4.12 (d, JH,H = 7.6 Hz, 1 H, 1ЈЈ-H),
2
8.0 Hz, 1 H, 5-H), 4.94 (d, JH,H = 6.8 Hz, 1 H, OCH2O), 4.89 (d,
4.04 (dd, 3JH,H = 5.4, 2JH,H = 11.6 Hz, 1 H, 5aЈЈ-H), 3.96 (dd, 3JH,H
3
2JH,H = 6.8 Hz, 1 H, OCH2O), 4.64 (d, JH,H = 5.3 Hz, 1 H, 1ЈЈ-
2
2
= 3.2, JH,H = 10.7 Hz, 1 H, 5aЈ-H), 3.89 (dd, 3JH,H = 4.6, JH,H
=
H), 4.26 (br., 3 H, 2Ј-H, 3Ј-H, 4Ј-H), 4.14 (br., 1 H, 3ЈЈ-H), 4.01
10.7 Hz, 1 H, 5bЈ-H), 3.57 (s, 3 H, OMe), 3.55–3.50 (m, 1 H, 4ЈЈ-
2
2
(d, JH,H = 10.8 Hz, 1 H, 5aЈ-H), 3.94 (d, JH,H = 10.8 Hz, 1 H,
H), 3.30 (dd, 3JH,H = 7.6, 3JH,H = 9.4 Hz, 1 H, 2ЈЈ-H), 3.25 (t, 3JH,H
3
2
5bЈ-H), 3.89 (dd, JH,H = 3.3, JH,H = 11.6 Hz, 1 H, 5aЈЈ-H), 3.78
3
= 9.4 Hz, 1 H, 3ЈЈ-H), 3.16 (t, JH,H = 11.6 Hz, 1 H, 5bЈЈ-H), 1.59
3
2
(dd, JH,H = 7.3, JH,H = 11.6 Hz, 1 H, 5bЈЈ-H), 3.69–3.68 (m, 1
H, 4ЈЈ-H), 3.55 (br., 1 H, 2ЈЈ-H), 3.48 (s, 3 H, OMe) ppm. 13C NMR
(67.8 MHz, CDCl3): δ = 164.2, 151.3, 140.4, 102.3, 100.3, 95.9,
89.9, 83.1, 74.9, 73.7, 70.1, 67.7, 61.1, 57.5, 56.3 ppm. HRMS
(ESI): calcd. for C16H22N8NaO9 [M + Na]+ 493.1408; found
493.1372.
(s, 3 H, CH3), 1.36 (s, 3 H, CH3) ppm. 13C NMR (67.8 MHz,
CDCl3): δ = 163.4, 150.0, 141.5, 114.4, 103.7, 102.2, 96.3, 93.2,
85.6, 84.7, 80.7, 78.2, 68.6, 65.5, 63.8, 61.0, 57.2, 27.1, 25.3 ppm.
HRMS (ESI): calcd. for C19H26N8NaO9 [M + Na]+ 533.1721;
found 533.1758.
2Ј,3Ј-O-Isopropylidene-5Ј-[(methyl 2,4-diazido-2,4-dideoxy-β-D-ri-
5Ј-[(Methyl 2,4-diamino-2,4-dideoxy-β-D-xylopyranosid-3-O-yl)-
bopyranosid-3-O-yl)methyl]uridine (23): MeOTf (12.5 µL,
0.110 mmol) was added to a stirred mixture of 20 (28.3 mg,
0.1032 mmol), 2Ј,3Ј-O-isopropylideneuridine (21; 42.2 mg,
0.148 mmol) and 4 A MS (0.2 g) in CH2Cl2 (1 mL) at room temp.
After 21 h, further MeOTf (12.5 µL, 0.110 mmol) was added. After
25 h from the start of the reaction, the reaction mixture was heated
to reflux. After 5 h at reflux, triethylamine (30 µL) was added and
the mixture was filtered through Celite and the solvents evaporated.
The residue was purified by chromatography on silica gel (hexane/
EtOAc, 3:1–1:1) to give 23 (36.4 mg, 69%) as crystals. Rf = 0.15
(hexane/EtOAc, 1:1); m.p. 149–150 °C. [α]2D5 = –41.4 (c = 0.432,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 9.43 (br., 1 H, NH),
methyl]uridine (7): Triphenylphosphane (150 mg, 0.572 mmol) was
added to a stirred solution of 24 (38.2 mg, 0.081 mmol) in pyridine
(1 mL) at 50 °C. After 16 h, a 28 % NH3 solution (1 mL) was
added. After 36 h from the start of the reaction, the solution was
evaporated and partitioned between water and CHCl3. The water
layer was evaporated and the residue was purified by chromatog-
raphy on silica gel (Kanto Co., 60N spherical gel, iPrOH/H2O/
28%NH3, 35:3:1–25:3:1–15:3:1) to give 7 (19.6 mg, 58%) as a white
solid. Rf = 0.30 (iPrOH/H2O/28%NH3, 10:3:1); m.p. 108–110 °C.
[α]2D5 = –8.6 (c = 0.444, H2O). H NMR (600 MHz, D2O): δ = 7.72
1
3
3
(d, JH,H = 8.1 Hz, 1 H, 6-H), 5.87 (d, JH,H = 8.1 Hz, 1 H, 5-H),
3
5.86 (d, JH,H = 3.3 Hz, 1 H, 1Ј-H), 4.97 (s, 2 H, OCH2O), 4.36 (t,
3
3
3JH,H = 3.3 Hz, 1 H, 2Ј-H), 4.23–4.21 (m, 3 H, 3Ј-H, 4Ј-H, 1ЈЈ-H),
7.40 (d, JH,H = 8.1 Hz, 1 H, 6-H), 5.77 (d, JH,H = 2.1 Hz, 1 H,
3
2
3
2
1Ј-H), 5.73 (d, JH,H = 8.1 Hz, 1 H, 5-H), 4.92–4.85 (m, 4 H, 2Ј-
4.00 (d, JH,H = 11.7 Hz, 1 H, 5aЈ-H), 3.96 (dd, JH,H = 4.9, JH,H
= 11.9 Hz, 1 H, 5aЈЈ-H), 3.90 (dd, JH,H = 3.8, JH,H = 11.7 Hz, 1
H, 5bЈ-H), 3.51 (s, 3 H, OMe), 3.33–3.25 (m, 2 H, 3ЈЈ-H, 5bЈЈ-H),
3
3
2
H, 3Ј-H, CH2), 4.63 (d, JH,H = 5.3 Hz, 1 H, 1ЈЈ-H), 4.38–4.34 (m,
3
3
1 H, 4Ј-H), 4.15 (t, JH,H = 3.3 Hz, 1 H, 3ЈЈ-H), 3.95 (dd, JH,H
=
=
2
3
2
3
3
3
3.7, JH,H = 10.7 Hz, 1 H, 5aЈ-H), 3.91 (dd, JH,H = 5.2, JH,H
2.91 (dt, JH,H = 4.9, JH,H = 9.8 Hz, 1 H, 4ЈЈ-H), 2.65 (t, JH,H
=
3
2
8.7 Hz, 1 H, 2ЈЈ-H) ppm. 13C NMR (67.8 MHz, D2O): δ = 169.9,
154.4, 142.1, 105.6, 103.1, 98.3, 90.6, 87.2, 82.7, 74.0, 70.2, 68.9,
66.6, 57.9, 56.5, 51.8 ppm. HRMS (ESI): calcd. for C16H27N4O9
[M + H]+ 419.1779; found 419.1780.
10.7 Hz, 1 H, 5bЈ-H), 3.87 (dd, JH,H = 3.8, JH,H = 11.6 Hz, 1 H,
5aЈЈ-H), 3.76 (dd, 3JH,H = 7.1, 2JH,H = 11.6 Hz, 1 H, 5bЈЈ-H), 3.69–
3
3
3.65 (m, 1 H, 4ЈЈ-H), 3.55 (dd, JH,H = 5.3, JH,H = 3.3 Hz, 1 H,
2ЈЈ-H), 3.48 (s, 3 H, OMe), 1.58 (s, 3 H, CH3), 1.36 (s, 3 H,
CH3) ppm. 13C NMR (150 MHz, CDCl3): δ = 163.3, 150.0, 141.7,
114.5, 102.3, 100.4, 95.8, 93.7, 85.8, 84.6, 80.87, 73.6, 68.6, 61.3,
61.2, 57.6, 56.3, 27.1, 25.3 ppm. HRMS (ESI): calcd. for
C19H26N8NaO9 [M + Na]+ 533.1721; found 533.1718.
1
Compound 7 (16 m
M
) + 2.0 Equiv. Zn(OAc)2: H NMR (600 MHz,
3
50 m [D3]AcONa buffer, pH 7.0, 30 °C): δ = 7.79 (d, JH,H
=
7.9 Hz, 1 H, 6-H), 5.95 (m, 2 H, 5-H, 1Ј-H), 4.95 (d, 2JH,H = 7.0 Hz,
2
1 H, OCH2O), 4.92 (d, JH,H = 7.0 Hz, 1 H, OCH2O), 4.72 (br., 1
H, 1ЈЈ-H), 4.44 (br., 1 H, 2Ј-H), 4.30–4.26 (m, 3 H, 3Ј-H, 4Ј-H,
5Ј-[(Methyl 2,4-diazido-2,4-dideoxy-β-D-xylopyranosid-3-O-yl)meth-
2
3
5aЈЈ-H), 4.01 (d, JH,H = 11.5 Hz, 1 H, 5aЈ-H), 3.94 (dd, JH,H
=
yl]uridine (24): A solution of 22 (72.1 mg, 0.141 mmol) in 80% ace-
tic acid (1.5 mL) was stirred for 21 h at 70 °C. After evaporation
of the solvent, the residue was purified by chromatography on silica
gel (CHCl3/MeOH, 20:1–15:1) to give 24 (52.9 mg, 80%) as a foam.
Rf = 0.29 (CHCl3/MeOH, 10:1). [α]2D5 = –16.0 (c = 1.00, MeOH).
2
4.2, JH,H = 11.5 Hz, 1 H, 5bЈ-H), 3.87 (br., 1 H, 3ЈЈ-H), 3.65 (d,
2JH,H = 12.5 Hz, 1 H, 5bЈЈ-H), 3.51 (s, 3 H, OMe), 3.37 (br., 1 H,
4ЈЈ-H), 3.33 (br., 1 H, 2ЈЈ-H), 1.98 (s, 12 H, Ac) ppm.
1
Compound 7 (16 mM) + 2.0 Equiv. Zn(OAc)2: H NMR (600 MHz,
3
1H NMR (400 MHz, [D6]DMSO): δ = 11.34 (br., 1 H, NH), 7.68 50 m [D3]AcONa buffer, pH 7.0, 70 °C): δ = 8.15 (d, JH,H
=
1604 Eur. J. Org. Chem. 2009, 1598–1605
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

A Novel Galactosyltransferase Inhibitor
2
8.0 Hz, 1 H, 6-H), 6.36 (d, 3JH,H = 8.0 Hz, 1 H, 5-H), 6.32 (d, 3JH,H
= 5.4 Hz, 1 H, 1Ј-H), 5.41 (d, JH,H = 7.0 Hz, 1 H, OCH2O), 5.38
2
2
= 4.4 Hz, 1 H, 1Ј-H), 5.35 (d, JH,H = 7.2 Hz, 1 H, OCH2O), 5.33
(d, JH,H = 7.0 Hz, 1 H, OCH2O), 5.29 (s, 1 H, 1ЈЈ-H), 4.84 (t,
(d, JH,H = 7.2 Hz, 1 H, OCH2O), 5.08 (br. s, 1 H, 1ЈЈ-H), 4.84 (t, 3JH,H = 5.4 Hz, 1 H, 2Ј-H), 4.71 (t, JH,H = 5.4 Hz, 1 H, 3Ј-H),
2
3
3JH,H = 4.4 Hz, 1 H, 2Ј-H), 4.69–4.64 (m, 3 H, 3Ј-H, 4Ј-H, 5aЈЈ- 4.67 (m, 1 H, 4Ј-H), 4.57 (t, JH,H = 4.4 Hz, 1 H, 3ЈЈ-H), 4.48 (m,
3
2
3
3
2
H), 4.41 (d, JH,H = 11.4 Hz, 1 H, 5aЈ-H), 4.33 (dd, JH,H = 4.9,
2 H, 5aЈ-H, 5aЈЈ-H), 4.32 (dd, JH,H = 4.7, JH,H = 11.6 Hz, 1 H,
2JH,H = 11.4 Hz, 1 H, 5bЈ-H), 4.21 (br. t, JH,H = 3.2 Hz, 1 H, 3ЈЈ- 5bЈ-H), 4.23 (d, JH,H = 13.0 Hz, 1 H, 5bЈЈ-H), 4.03 (m, 1 H, 4ЈЈ-
3
2
3
2
3
H), 4.03 (dd, JH,H = 3.4, JH,H = 12.8 Hz, 1 H, 5bЈ-H), 3.92 (s, 3
H, OMe), 3.75 (br., 1 H, 4ЈЈ-H), 3.68 (br., 1 H, 2ЈЈ-H), 2.39 (s, 12
H, Ac) ppm.
H), 3.96 (d, JH,H = 4.2 Hz, 1 H, 2ЈЈ-H), 3.87 (s, 3 H, OMe), 2.39
(s, 12 H, Ac) ppm.
Supporting Information (see also the footnote on the first page of
this article): Details of assay methods and H and 13C NMR spec-
1
5Ј-[(Methyl 2,4-diamino-2,4-dideoxy-β-D-ribopyranosid-3-O-yl)-
tra of the compounds.
methyl]uridine (8): Triphenylphosphane (312.5 mg, 1.19 mmol) was
added to a stirred solution of 25 (76.7 mg, 0.163 mmol) in pyridine
(2 mL) at 50 °C. After 16 h, a 28% NH3 solution (2 mL) was
added. After 21 h from the start of the reaction, the solution was
evaporated and partitioned between water and CHCl3. The water
layer was evaporated and the residue was purified by chromatog-
raphy on silica gel (Kanto Co., 60N spherical gel, iPrOH/H2O/
28%NH3, 35:3:1–25:3:1–15:3:1) to give 8 (46.6 mg, 68%) as a white
solid. Rf = 0.18 (iPrOH/H2O/28%NH3, 10:3:1); m.p. 104–105 °C.
Acknowledgments
This work was supported by a grant from the Global COE Program
and a Grant-in-Aid for Scientific Research (No. 19655056) from
the Japanese Ministry of Education, Culture, Sports, Science and
Technology.
[α]2D5 = –23.1 (c = 0.54, H2O). H NMR (600 MHz, D2O): δ = 7.74
1
3
3
[1] C.-H. Wong, R. L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew.
Chem. Int. Ed. Engl. 1995, 34, 521–546.
(d, JH,H = 8.1 Hz, 1 H, 6-H), 5.87 (d, JH,H = 5.0 Hz, 1 H, 1Ј-H),
3
2
5.85 (d, JH,H = 8.1 Hz, 1 H, 5-H), 4.93 (d, JH,H = 7.1 Hz, 1 H,
OCH2O), 4.90 (d, JH,H = 7.1 Hz, 1 H, OCH2O), 4.52 (d, JH,H
[2] Y. Shiro, K. Kato, M. Fujii, Y. Ida, H. Akita, Tetrahedron 2006,
2
3
=
62, 8687–8695.
3
5.8 Hz, 1 H, 1ЈЈ-H), 4.35 (t, JH,H = 5.0 Hz, 1 H, 2Ј-H), 4.23 (t,
[3] P. Compain, O. R. Martin, Bioorg. Med. Chem. 2001, 9, 3077–
3092.
3JH,H = 5.0 Hz, 1 H, 3Ј-H), 4.21–4.19 (m, 1 H, 4Ј-H), 4.00 (dd,
3JH,H = 2.2, JH,H = 11.5 Hz, 1 H, 5aЈ-H), 3.96 (br., 1 H, 3ЈЈ-H),
2
[4] M. M. Vaghefi, R. J. Bernacki, W. J. Hennen, R. K. Robins, J.
Med. Chem. 1987, 30, 1391–1399.
3.88 (dd, 3JH,H = 5.0, 2JH,H = 11.5 Hz, 1 H, 5bЈ-H), 3.79 (dd, 3JH,H
2
3
2
= 3.5, JH,H = 11.7 Hz, 1 H, 5aЈЈ-H), 3.61 (dd, JH,H = 8.3, JH,H
= 11.7 Hz, 1 H, 5bЈЈ-H), 3.44 (s, 3 H, OMe), 3.18 (br., 1 H, 4ЈЈ-H),
2.90 (br., 1 H, 2ЈЈ-H) ppm. 13C NMR (67.8 MHz, D2O): δ = 168.9,
153.7, 142.4, 103.0, 97.4, 90.7, 82.9, 78.1, 74.0, 70.2, 68.6, 64.3,
57.1, 53.8, 49.8 ppm. HRMS (ESI): calcd. for C16H27N4O9 [M +
H]+ 419.1779; found 419.1741.
[5] M. E. Colvin, E. Evleth, Y. Akacem, J. Am. Chem. Soc. 1995,
117, 4357–4362.
[6] S. Murata, S. Ichikawa, A. Matsuda, Tetrahedron 2005, 61,
5837–5842.
[7] R. Wang, D. H. Steensma, Y. Takaoka, J. W. Yun, T. Kajimoto,
C.-H. Wong, Bioorg. Med. Chem. 1997, 5, 661–672.
[8] L. Ballell, R. J. Young, R. A. Field, Org. Biomol. Chem. 2005,
3, 1109–1115.
[9] H. Yuasa, T. Izumi, N. Mitsuhashi, Y. Kajihara, H. Hashim-
oto, Chem. Eur. J. 2005, 11, 6478–6490.
[10] H. Yuasa, N. Miyagawa, M. Nakatani, M. Izumi, H. Hashim-
oto, Org. Biomol. Chem. 2004, 2, 3548–3556.
[11] X. Liu, C. B. Reese, J. Chem. Soc. Perkin Trans. 1 2000, 2227–
2236.
1
Compound 8 (16 m
M
) + 2.0 Equiv. Zn(OAc)2: H NMR (600 MHz,
3
50 m [D3]AcONa buffer, pH 7.0, 30 °C): δ = 7.76 (d, JH,H
=
3
8.3 Hz, 1 H, 6-H), 5.87 (d, JH,H = 5.3 Hz, 1 H, 1Ј-H), 5.66 (d,
3JH,H = 8.3 Hz, 1 H, 5-H), 4.92 (d, JH,H = 7.1 Hz, 1 H, OCH2O),
2
2
4.87 (d, JH,H = 7.1 Hz, 1 H, OCH2O), 4.79 (s, 1 H, 1ЈЈ-H), 4.35
(t, JH,H = 5.3 Hz, 1 H, 2Ј-H), 4.25 (t, JH,H = 5.3 Hz, 1 H, 3Ј-H),
4.19 (br., 1 H, 4Ј-H), 4.09 (br. s, 1 H, 3ЈЈ-H), 4.01–3.97 (m, 2 H,
3
3
[12] The 4C1/1C4 ratios were calculated by a multiple regression
analysis with a least-squares fitting of the J values calculated
for model structures to observed values. For details, see ref.[9]
[13] J. T. Powell, K. Brew, Biochemistry 1976, 15, 3499–3505.
[14] D. D. Perrin, W. L. Armarego, D. R. Perrin, Purification of
Laboratory Compounds, 2nd ed., Pergamon Press, London,
1980.
3
2
5aЈ-H, 5aЈЈ-H), 3.81 (dd, JH,H = 3.3, JH,H = 11.2 Hz, 1 H, 5bЈ-
H), 3.72 (d, 2JH,H = 13.0 Hz, 1 H, 5bЈЈ-H), 3.54 (br. s, 1 H, 4ЈЈ-H),
3
3.44 (d, JH,H = 3.3 Hz, 1 H, 2ЈЈ-H), 3.36 (s, 3 H, OMe), 1.89 (s,
12 H, Ac) ppm.
1
Compound 8 (16 m
M
) + 2.0 Equiv. Zn(OAc)2: H NMR (600 MHz,
3
50 m [D3]AcONa buffer, pH 7.0, 70 °C): δ = 8.19 (d, JH,H
=
Received: December 26, 2008
Published Online: February 18, 2009
8.1 Hz, 1 H, 6-H), 6.35 (d, 3JH,H = 8.1 Hz, 1 H, 5-H), 6.34 (d, 3JH,H
Eur. J. Org. Chem. 2009, 1598–1605
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1605