Synthesis of bisubstrate and donor analogues of sialyltransferase and their inhibitory activities

Article abstract of DOI:10.1021/jo0512608

Sialyltransferases (STs) are involved in the biosynthesis of glycoconjugates with important biological activities. Most STs utilize cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) as a common donor substrate. A bisubstrate analogue containing the donor substrate (CMP-Neu5Ac mimic) and the acceptor substrate (galactose) was synthesized. Four donor analogues having the partial structure of the bisubstrate analogue were also synthesized to support study of the structure-activity relationship. Each analogue contains an ethylene group in place of the exocyclic anomeric oxygen of CMP-Neu5Ac. The bisubstrate analogue exhibited only weak inhibitory activity to rat recombinant α-2,3- and α-2,6-ST (IC₅₀ = 1.3, 2.4 mM). Conversion of the C-1 carboxylate of the Neu5Ac moiety to carboxyamide, hydroxymethyl, or methylene phosphate each resulted in a reduction in inhibitory activity. Among the synthesized analogues, cytidin-5′-yl sialylethylphosphonate (4) was the most potent inhibitor against rat recombinant α-2,3- and α-2,6-ST (IC₅₀ = 0.047, 0.34 mM).

Full text of DOI:10.1021/jo0512608

Synthesis of Bisubstrate and Donor Analogues of Sialyltransferase
and Their Inhibitory Activities
Masayuki Izumi,* Katsuhiro Wada, Hideya Yuasa, and Hironobu Hashimoto
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of
Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
masayuki.izumi@aist.go.jp
Received June 20, 2005
Sialyltransferases (STs) are involved in the biosynthesis of glycoconjugates with important biological
activities. Most STs utilize cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) as
a common donor substrate. A bisubstrate analogue containing the donor substrate (CMP-Neu5Ac
mimic) and the acceptor substrate (galactose) was synthesized. Four donor analogues having the
partial structure of the bisubstrate analogue were also synthesized to support study of the
structure-activity relationship. Each analogue contains an ethylene group in place of the exocyclic
anomeric oxygen of CMP-Neu5Ac. The bisubstrate analogue exhibited only weak inhibitory activity
to rat recombinant R-2,3- and R-2,6-ST (IC50 ) 1.3, 2.4 mM). Conversion of the C-1 carboxylate of
the Neu5Ac moiety to carboxyamide, hydroxymethyl, or methylene phosphate each resulted in a
reduction in inhibitory activity. Among the synthesized analogues, cytidin-5′-yl sialylethylphos-
phonate (4) was the most potent inhibitor against rat recombinant R-2,3- and R-2,6-ST (IC50
0.047, 0.34 mM).
)
Introduction
Most STs utilize cytidine-5′-monophospho-N-acetyl-
neuraminic acid (CMP-Neu5Ac 1, Figure 1) as a glycosyl
donor. We have been studying the synthesis of stable
CMP-Neu5Ac analogues as potential sialyltransferase
inhibitors and have reported the synthesis of 25 and 3,6
in which the glycosidic oxygen has been removed and
substituted with a methylene group, respectively. The
Schmidt group also synthesized 2, and the Ki value
against rat recombinant R-2,6-ST was reported to be 0.25
mM.7 Several stable donor analogues of glycosyltrans-
ferases having glycosyl phosphonate, glycosyl C-alkyl-
phosphonate, and glycosyl C-alkyl phosphate have also
been synthesized.8,9 Uridine-5′-diphosphogalactose (UDP-
Gal) analogues were tested as â-1,4-galactosyltransferase
inhibitors and demonstrated Ki values similar to the KM
value of UDP-Gal.9 This suggests that glycosyltrans-
ferases can tolerate this type of structural modification.
The Schmidt group has extensively studied sialyltrans-
ferase inhibitors10 based on CMP-Neu5Ac and its oxo-
Sialic acids, such as N-acetylneuraminic acid (Neu5Ac),
are often located at the nonreducing end of oligosaccha-
rides in glycoproteins and glycolipids.1 Sialic acids are
also involved in various biological events, such as bacter-
ial or viral infection, inflammation, development, and
tumor progression.2 Sialyltransferases (STs) are the
enzymes responsible for biosyntheses of the sialic acid-
containing oligosaccharides.3 Sialyltransferase inhibitors4
are thus useful biological probes for studying sialic acid-
mediated biological processes. These inhibitors are also
valuable for exploring the active sites of STs and for
elucidating the mechanisms of action of these enzymes.
* Current address of corresponding author: Research Center of
Advanced Bionics, National Institute of Advanced Industrial Science
and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565,
Japan. Phone: +81-29-861-4681. Fax: +81-29-861-4680.
(1) Varki, A. In Essentials of Glycobiology; Varki, A., Cummings,
R., Esko, J., Freeze, H., Hart, G., Marth, J., Eds.; Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, 1999; p 195-209.
(2) Varki, A. Glycobiology 1993, 3, 97-130.
(3) Lau, J. T. Y.; Wuensch, S. A. In Carbohydrates in Chemistry and
Biology; Ernst, B., Hart, G. W., Sina¨y, P., Eds.; Wiley-VCH: Weinheim,
2000; Vol. 3, p 213-226.
(4) Wang, X.; Zhang, L.-H.; Ye, X.-S. Med. Res. Rev. 2003, 23, 32-
47.
(5) Imamura, M.; Hashimoto, H. Tetrahedron Lett. 1996, 37, 1451-
1454.
(6) Imamura, M.; Hashimoto, H. Chem. Lett. 1996, 1087-1088.
(7) Muller, B.; Martin, T. J.; Schaub, C.; Schmidt, R. R. Tetrahedron
Lett. 1998, 39, 509-512.
10.1021/jo0512608 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/30/2005
J. Org. Chem. 2005, 70, 8817-8824
8817

Izumi et al.
GalT. Bisubstrate analogue inhibitors have also been
reported for fucosyltransferases.15 For sialyltransferases,
the Schmidt group synthesized bisubstrate analogue
inhibitors by adding acceptor analogues to their transi-
tion state inhibitors but reported that neither the result-
ing inhibitory potency nor the selectivity between rat
recombinant R-2,3- and R-2,6-STs was increased.10 Bisub-
strate analogue inhibitors reported by Hinou et al. were
also good inhibitors of both rat recombinant R-2,3- and
R-2,6-STs, with Ki values similar to the KM value of
CMP-Neu5Ac.16 This bisubstrate analogue strategy for
the development of potent and selective glycosyltrans-
ferase inhibitors may be effective only for inverting
enzymes such as sialyltransferases, â-galactosyltrans-
ferases, and fucosyltransferases, as retaining enzymes
are assumed to transfer a glycosyl moiety via a glycosyl-
enzyme intermediate.17
In our continuing synthetic studies of stable CMP-
Neu5Ac analogues, we planned the synthesis of cytidin-
5′-yl sialylethylphosphonate (4, Figure 1), which pos-
sesses an ethylene group rather than a glycosidic oxygen
and resembles the transition state of glycosyl transfer,
by moving CMP one bond farther from the Neu5Ac
residue. We expanded our studies to include bisubstrate
analogue 8, which contains the partial structure of donor
analogue 4, and methyl â-galactoside as an acceptor
analogue. Taking into consideration that R-glycoside is
formed by STs, we placed an acceptor moiety at the R-face
of the Neu5Ac residue via the C-1 hydroxymethyl group,
which was formed via reduction of carboxylate. The donor
and the acceptor analogues were linked by a phosphate
group, which also mimics the negative charge of C-1
carboxylate. In this study, methyl â-galactoside was used
as an acceptor analogue rather than the high-affinity
acceptor LacNAc,18 in order to simplify the synthesis.
Donor analogues (6, 7) possessing the partial structure
of 8 were also prepared to evaluate our inhibitor design.
Here, we report the synthesis of donor analogues 4-7,
as well as the bisubstrate analogue 8, and their inhibitory
activities toward rat recombinant R-2,3- and R-2,6-
sialyltransferases.
FIGURE 1. CMP-Neu5Ac and designed analogues.
carbenium transition state11 in ST-catalyzed reactions.
From these studies, they revealed that R-hydroxyphos-
phonate esters of CMP, having a flattened ring system
resembling the transition state of glycosyl transfer, are
very strong sialyltransferase inhibitors, having Ki values
in the nanomolar range.12 The most potent inhibitor
reported by the group has a Ki value of 29 nM, which is
roughly 4 orders of magnitude smaller than the KM value
of CMP-Neu5Ac (47 µM).13
There are three typical types of glycosidic linkages
found in sialic acids: R-2,3-galactose (Gal); R-2,6-Gal,
N-acetylgalactosamine, or N-acetylglucosamine (GlcNAc);
and R-2,8-Neu5Ac. Because most STs catalyze the trans-
fer of Neu5Ac from CMP-Neu5Ac, the specificity of STs
most likely results from recognition of the glycosyl
acceptor. Therefore, bisubstrate analogues in which a
donor and an acceptor analogue are covalently connected,
enabling interaction with both substrate-binding sites,
are candidates for selective ST inhibitors. We previously
reported that a bisubstrate analogue composed of UDP-
Gal (donor) and GlcNAc (acceptor), connected via a
methylene group, was a potent â-1,4-galactosyltrans-
ferase (GalT) inhibitor14 but was not an inhibitor of R-1,3-
Results and Discussion
For the convergent synthesis of all analogues, nonitol
15, which contains a protected hydroxymethyl group ra-
ther than an ester, was designed as a common intermedi-
ate. The synthesis of 15 is illustrated in Scheme 1. All
the hydroxyl groups of â-C-allyl sialoside (9)19 were
protected with benzyloxymethyl (BOM) groups, and re-
duction of the methyl ester with LiEt3BH yielded alcohol
11. The hydroxymethyl group in 11 was protected with
(8) (a) Stolz, F.; Blume, A.; Hinderlich, S.; Reutter, W.; Schmidt, R.
R. Eur. J. Org. Chem. 2004, 3304-3312. (b) Schafer, A.; Thiem, J. J.
Org. Chem. 2000, 65, 24-29. (c) Luengo, J. I.; Gleason, J. G.
Tetrahedron Lett. 1992, 33, 6911-6914. (d) Orbe, M.; Claesson, A. Eur.
J. Med. Chem. 1989, 24, 447-451. (e) Norbeck, D. W.; Kramer, J. B.;
Lartey, P. A. J. Org. Chem. 1987, 52, 2174-2179.
(9) (a) Schmidt, R. R.; Frische, K. Bioorg. Med. Chem. Lett. 1993, 3,
1747-1750. (b) Vaghefi, M. M.; Bernacki, R. J.; Dalley, N. K.; Wilson,
B. E.; Robins, R. K. J. Med. Chem. 1987, 30, 1383-1391.
(10) Jung, K.-H.; Schwoerer, R.; Schmidt, R. R. Trends Glycosci.
Glycotechnol. 2003, 15, 275-289.
(11) (a) Bruner, M.; Horenstein, B. A. Biochemistry 2000, 39, 2261-
8. (b) Bruner, M.; Horenstein, B. A. Biochemistry 1998, 37, 289-97.
(12) Muller, B.; Schaub, C.; Schmidt, R. R. Angew. Chem., Int. Ed.
1998, 37, 2893-2897.
(13) Schwoerer, R.; Schmidt, R. R. J. Am. Chem. Soc. 2002, 124,
1632-1637.
(14) Hashimoto, H.; Endo, T.; Kajihara, Y. J. Org. Chem. 1997, 62,
1914-1915.
(15) (a) Palcic, M. M.; Heerze, L. D.; Srivastava, O. P.; Hindsgaul,
O. J. Biol. Chem. 1989, 264, 17174-17181. (b) Qiao, L.; Murray, B.
W.; Shimazaki, M.; Schultz, J.; Wong, C.-H. J. Am. Chem. Soc. 1996,
118, 7653-7662. (c) Jefferies, I.; Bowen, B. R. Bioorg. Med. Chem. Lett.
1997, 7, 1171-1174. (d) Pasquarello, C.; Picasso, S.; Demange, R.;
Malissard, M.; Berger, E. G.; Vogel, P. J. Org. Chem. 2000, 65, 4251-
4260.
(16) Hinou, H.; Sun, X.-L.; Ito, Y. J. Org. Chem. 2003, 68, 5602-
5613.
(17) Unligil, U. M.; Rini, J. M. Curr. Opin. Struct. Biol. 2000, 10,
510-517.
(18) Williams, M. A.; Kitagawa, H.; Datta, A. K.; Paulson, J. C.;
Jamieson, J. C. Glycoconjugate J. 1995, 12, 755-761.
(19) Paulsen, H.; Matschulat, P. Liebigs Ann. Chem. 1991, 487-
495.
8818 J. Org. Chem., Vol. 70, No. 22, 2005

Bisubstrate and Donor Analogues of Sialyltransferase
SCHEME 1a
SCHEME 2a
a Reagents and conditions: (a) BOMCl, DIEA, DMF, 60 °C, 93%;
(b) LiEt3BH, THF, 98%; (c) TBDMSCl, imidazole, DMF, 93%; (d)
O3, MeOH, CH2Cl2, -78 °C then Me2S, 92%; (e) BuLi, (MeO)2POH,
THF, -78 °C then ClC(S)OPh, 92%; (f) Bu3SnH, AIBN, toluene,
70 °C, 75%; (g) Pd-C, HCO2NH4, MeOH, reflux; (h) Ac2O, pyridine,
DMAP, 87% (two steps)
a Reagents and conditions: (a) HF-pyridine, THF, 80%; (b)
RuCl3‚nH2O, NaIO4, CCl4, MeCN, H2O, 93%; (c) CH2N2, Et2O,
benzene, EtOH, 81%; (d) PhSH, Et3N, dioxane, 87%; (e) BOP,
DIEA, DMF; (f) PhSH, Et3N, dioxane, 73% from 19; (g) NH4OH,
MeOH (1:1), 4, 32%, 5, 30%
a tert-butyldimethylsilyl (TBDMS) group to yield 12,
which was then converted to aldehyde 13 by ozonolysis.
Addition of dimethyl phosphite [(MeO)2POH] to 13 with
BuLi and subsequent phenoxythiocarbonylation of the
newly formed hydroxyl group20 yielded R-(phenoxythio-
carbonyloxy)ethylphosphonate in a diastereomeric mix-
ture. Deoxygenation with Bu3SnH yielded nonitolyleth-
ylphosphonate 14. BOM groups were removed by hydro-
genation, and subsequent acetylation yielded common
intermediate 15.
SCHEME 3a
Intermediate 15 was next converted to 4 and 5 (Scheme
2). Treatment of 15 with HF-pyridine yielded alcohol 16.
Oxidation of the primary alcohol with ruthenium tetrox-
ide21 yielded carboxylic acid 17, which was converted to
methyl ester 18 via treatment with diazomethane.
Chemoselective demethylation of ester 18, using thiophen-
ol (PhSH) and triethylamine (Et3N) in dioxane22 yielded
monomethyl phosphonate 19. Condensation of monoester
19 and triacetylcytidine (20)23 using benzotriazol-1-
yloxytris(dimethyamino)phosphonium hexafluorophos-
phate (BOP) and N,N-diisopropylethylamine (DIEA) in
DMF24 yielded the fully protected cytidin-5′-yl sialyleth-
ylphosphonate with very good yield. The methyl group
of the phosphonate was removed with PhSH and Et3N
to give 21. Simultaneous deacetylation and saponification
using ammonium hydroxide (NH4OH) and methanol
(MeOH) resulted in the formation of carboxylic acid 4 and
carboxyamide 5 in a 1:1 ratio. Monovalent anion 5 was
eluted more rapidly from an anion-exchange column than
a Reagents and conditions: (a) PhSH, Et3N, dioxane, 86%; (b)
BOP, DIEA, DMF; (c) PhSH, Et3N, dioxane; (d) THF, HCO2H, H2O
(6:3:1); (e) NH4OH, MeOH (1:1), 57% from 22
(20) Martin, S. F.; Dean, D. W.; Wagman, A. S. Tetrahedron Lett.
1992, 33, 1839-1842.
divalent anion 4, and the structures of 4 and 5 were
elucidated by ESI-MS analysis.
Hydroxymethyl derivative 6 was next synthesized
(Scheme 3). Demethylation of common intermediate 15,
using PhSH and Et3N, yielded monoester 22. Condensa-
tion of 22 with 20, using BOP/DIEA, yielded protected
(21) Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B.
J. Org. Chem. 1981, 46, 3936-3938.
(22) Daub, G. W.; Van Tameken, E. E. J. Am. Chem. Soc. 1977, 99,
3526-3528.
(23) Baschang, G.; Kvita, V. Angew. Chem. 1973, 85, 43-44.
(24) Campagne, J.-M.; Coste, J.; Jouin, P. J. Org. Chem. 1995, 60,
5214-5223.
J. Org. Chem, Vol. 70, No. 22, 2005 8819

Izumi et al.
SCHEME 4a
TABLE 1. Inhibitory Activities of Compounds 4-8
against Rat Recombinant r-2,3- and r-2,6-STs
a
IC50
compd
R-2,3-ST
R-2,6-ST
4
5
6
7
8
0.047
3.3
0.34
4.3
3.2
2.3
2.4
4.2
0.95
1.3
a All values are in mM. IC50 values at 0.11 mM CMP-Neu5Ac
and 1.0 mM PA-LacNAc.
pyridyl)amino]ethyl â-N-acetyllactosaminide (PA-Lac-
NAc), in which the reaction product was quantitated via
RP-HPLC.26 To evaluate inhibitory activities, IC50 values
were measured for all analogues under conditions for
both substrates (i.e. 0.11 mM for CMP-Neu5Ac and 1.0
mM for PA-LacNAc). Only carboxylate derivative 4
exhibited significant inhibitory activities against both
STs (IC50 ) 0.047 mM for R-2,3-ST and 0.34 mM for R-2,6-
ST). Since the difference between these two IC50 values
is about 1 order of magnitude, compound 4 could be a
good starting structure for the design of the selective
bisubstrate analogue inhibitor. Analogues 5 and 6, each
of which has only one negative charge, were poor inhibi-
tors, with IC50 values in the millimolar range. Replacing
the carboxylate in 4 with a methylene phosphate group
also resulted in reduced inhibitory activity, as observed
for 7 and 8. However, IC50 values of 7 and 8 were
relatively smaller than those of 5 and 6, suggesting the
importance of the existence of the second negative charge.
Schmidt et al. suggested that the distance between two
negative charges should be about five bonds, to result in
a strong inhibitory activity.10 The weak inhibitory activi-
ties of 7 and 8 may result from the two negative charges
being spaced too far apart. No improvement in the
inhibitory activity of bisubstrate analogue 8 suggested
that the galactose residue did not contribute to the
additional binding. However, our linking strategy of the
two substrate analogues through the 1-position of Neu5Ac
may be effective, since the inhibitory potency was not
interfered with by the bulky galactose residue, although
connecting the donor and the acceptor through phosphate
as a mimic of carboxylate was not effective. According to
the structure of Schmidt’s nanomolar inhibitors, the
presence of two negative charges, an extended distance
between the anomeric carbon and the CMP leaving
group, and an anomeric carbon with sp2 hybridization is
important.10 Recently, a crystallographic structure of the
sialyltransferase CstII from Campylobacter jejuni in
complex with CMP-3′′F-Neu5Ac was reported.27 In this
structure, the pyranose ring of Neu5Ac had the 0S5 skew
conformation, which also suggested the importance of the
flattened ring conformation for high-affinity binding. The
similar affinity of 4 and CMP-Neu5Ac is probably due
to the lack of an sp2 anomeric carbon.
a Reagents and conditions: (a) TBAF, THF, AcOH, 64%; (b)
(EtO)2PCl, DIEA, CH2Cl2; (c) tBuOOH, MeCN; (d) LiBr, MeCN,
reflux; (e) NH4OH, MeOH (1:1), 50% from 24; (f) 1H-tetrazole,
MeCN; (g) tBuOOH; (h) PhSH, Et3N, dioxane, 43% from 24; (i)
NH4OH, MeOH (1:1), 53%
cytidin-5′-yl nonitolylethylphosphonate 23, with good
yield. The methyl group was first removed by treatment
with PhSH and Et3N. The TBDMS group was then re-
moved smoothly under acidic conditions with anchimeric
assistance from the adjacent phosphonic acid.25 Removal
of all acetyl groups with NH4OH-MeOH yielded 6.
Finally, analogues having phosphate groups were
synthesized (Scheme 4). To release the free hydroxyl
group at the C-1 of the nonitol residue, the TBDMS group
of 23 was removed by treatment with tetrabutylammo-
nium fluoride (TBAF). It was necessary to use com-
mercially available 1 M TBAF in THF without dilution
to complete the deprotection, possibly due to steric
hindrance at the C-1 position. Phosphorylation of alcohol
24 with diethyl chlorophosphite and DIEA, followed by
oxidation with tert-butyl hydroperoxide, yielded the di-
ethyl phosphate. Treatment of the diethyl phosphate with
LiBr in refluxing MeCN, followed by deacetylation yielded
monoethyl phosphate 7. Alcohol 24 was also converted
into bisubstrate analogue 8 in the following manner.
Condensation of alcohol 24 and galactosid-6-yl phos-
phoramidite 25 with tetrazole and subsequent oxidation
yielded a fully protected bisubstrate analogue, which was
then treated with PhSH and Et3N to give phosphodiester
26 at a 43% yield, in three steps. Deacetylation of 26
yielded 8, which was purified by anion-exchange chro-
matography and gel permeation chromatography.
In conclusion, we synthesized five CMP-Neu5Ac ana-
logues, each of which has an ethylene group rather than
a glycosidic oxygen and one of which is a bisubstrate
analogue. An inhibition assay for two sialyltransferases
The inhibitory activities of CMP-Neu5Ac analogues
4-8 against rat recombinant R-2,3- and R-2,6-STs18 were
measured and are summarized in Table 1. The ST assay
was carried out using a fluorescent acceptor, 2-[(2-
(26) Kajihara, Y.; Kamiyama, D.; Yamamoto, N.; Sakakibara, T.;
Izumi, M.; Hashimoto, H. Carbohydr. Res. 2004, 339, 1545-1550.
(27) Chiu, C. P. C.; Watts, A. G.; Lairson, L. L.; Gilbert, M.; Lim,
D.; Wakarchuk, W. W.; Withers, S. G.; Strynadka, N. C. J. Nat. Struct.
Mol. Biol. 2004, 11, 163-170.
(25) Kawahara, S.-i.; Wada, T.; Sekine, M. J. Am. Chem. Soc. 1996,
118, 9461-9468.
8820 J. Org. Chem., Vol. 70, No. 22, 2005

Bisubstrate and Donor Analogues of Sialyltransferase
9H), 0.03, 0.02 (each s, 6H); 13C NMR (67.8 MHz, CDCl3) δ
170.2, 137.8, 137.7, 137.2, 133.1, 128.5, 128.3, 127.9, 127.4,
118.0, 97.0, 95.7, 95.0, 93.6, 78.3, 77.7, 77.1, 71.2, 70.6, 69.5,
69.3, 69.1, 68.4, 54.7, 37.3, 34.4, 25.7, 23.4, 18.0, -5.55, -5.60;
HR-ESI-MS calcd for C52H72NO11Si [M + H]+ 914.4875, found
914.4877.
revealed that replacing the carboxylate of Neu5Ac with
the methylene phosphate group resulted in a loss of
affinity. Although four of the five analogues failed to
exhibit substantial inhibitory activity against the STs,
cytidin-5′-yl sialylethylphosphonate (4) was a good in-
hibitor against both rat recombinant R-2,3 and R-2,6-ST.
The linking strategy and structure of the acceptor moiety
still need to be refined for a selective bisubstrate inhibitor
based on cytidin-5′-yl sialylethylphosphonate structure.
6-Acetamido-3,7-anhydro-5,8,9,10-tetra-O-benzyloxy-
methyl-3-C-tert-butyldimethylsilyloxymethyl-2,4,6-tri-
deoxy-D-erythro-L-manno-decose (13). Into a solution of 12
(1.345 g, 1.47 mmol) in MeOH (11.6 mL) and CH2Cl2 (2.9 mL)
was bubbled O3 for 20 min at -78 °C. Then, O2 gas was bubble
into the solution for 5 min at the same temperature. Me2S (2.3
mL) was added to the solution, and the mixture stirred for
1.5 h at the same temperature and then stirred overnight at
room temperature. The solution was concentrated and the
residue was purified on a column of silica gel (toluene-acetone
7:1) to yield 13 (1.246 g, 92%) as a faint yellow syrup: 1H NMR
(270 MHz, CDCl3) δ 9.68 (br, 1H), 7.41-7.24 (m, 20H), 5.76
(d, 1H, J ) 7.3 Hz), 4.98-4.50 (m, 17H), 4.41 (d, 1H, J ) 9.9
Hz), 4.01 (dd, 1H, J ) 2.0, 10.9 Hz), 3.98-3.89 (m, 2H), 3.79
(dd, 1H, J ) 3.6, 10.9 Hz), 3.68 (d, 1H, J ) 9.6 Hz), 3.55 (d,
1H, J ) 9.6 Hz), 3.33 (dt, 1H, J ) 7.3, 9.9 Hz), 2.89 (dd, 1H,
J ) 2.0, 15.2 Hz), 2.70 (dd, 1H, J ) 3.0, 15.2 Hz), 2.41 (dd,
1H, J ) 4.9, 11.9 Hz), 1.65 (s, 3H), 1.42 (t, 1H, J ) 11.9 Hz),
0.89-0.81 (s, 9H), 0.01 (s, 6H); 13C NMR (67.8 MHz, CDCl3) δ
199.6, 170.2, 137.60, 137.58, 137.0, 128.5, 128.3, 128.04,
127.96, 127.53, 127.51, 127.49, 97.2, 95.9, 95.2, 93.9, 77.7, 76.9,
70.93, 70.90, 70.1, 69.8, 69.6, 69.33, 69.31, 68.0, 54.8, 44.9, 38.3,
25.8, 23.7, 18.2, -5.4, -5.5; HR-ESI-MS calcd for C51H70NO12-
Si [M + H]+ 916.4667, found 916.4672.
Dimethyl 2-(5-Acetamido-2,6-anhydro-4,7,8,9-tetra-O-
benzyloxymethyl-1-O-tert-butyldimethylsilyl-3,5-dideoxy-
D-erythro-L-gluco-nonitol-2-C-yl)ethylphosphonate (14).
To a solution of dimethyl phosphite (76 µL, 0.83 mmol) in THF
(3.9 mL) was added dropwise a solution of n-BuLi (1.6 M
hexane solution, 0.52 mL, 0.83 mmol) at -78 °C under Ar
atmosphere. After the mixture stirred for 7 min, a solution of
13 (0.475 g, 0.518 mmol) in THF (1.35 mL) was added dropwise
to it. After the mixture stirred for 5 min at -78 °C, phenyl
thionochloroformate (0.28 mL, 2.1 mmol) was added. After
stirring for 30 min at the same temperature, saturated
aqueous NH4Cl was added. The aqueous layer was extracted
three times with CHCl3, and the combined organic layer was
dried (MgSO4) and concentrated. The residue was purified on
a column of silica gel (toluene-acetone 5:1f4:1) to yield
dimethyl 2-(5-acetamido-2,6-anhydro-4,7,8,9-tetra-O-benzyl-
oxymethyl-1-O-tert-butyldimethylsilyl-3,5-dideoxy-D-erythro-L-
gluco-nonitol-2-C-yl)-1-phenoxylthiocarbonyloxyethylphos-
phonate (0.519 g, 86%) as a faint yellow syrup: 31P NMR
(CDCl3) δ 22.16, 21.71.
A solution of n-Bu3SnH (1.37 g, 4.69 mmol) and AIBN (50
mg) in dry toluene (33.6 mL) was heated to 70 °C. A solution
of the yellow syrup (1.17 g, 1.01 mmol) in dry toluene (5.6 mL)
was added dropwise to the solution. After being stirred for 2
h at the same temperature, the solution was concentrated, and
the residue was purified on a column of silica gel (toluene-
acetone 4:1f3:1) to yield 14 (0.764 g, 75%) as a colorless
syrup: 1H NMR (400 MHz, CDCl3) δ 7.47 (m, 20H), 5.77 (d,
1H, J ) 7.9 Hz), 4.90-4.52 (m, 16H), 4.41 (dt, 1H, J ) 4.7,
12.5 Hz), 4.14 (d, 1H, J ) 10.5 Hz), 4.07 (dd, 1H, J ) 1.8, 9.8
Hz), 4.01-3.96 (m, 2H), 3.82 (dd, 1H, J ) 4.3, 10.5 Hz), 3.70,
3.69 (each d, 3H×2, J ) 10.0 Hz), 3.53-3.46 (m, 2H), 3.35 (d,
1H, J ) 9.6 Hz), 2.24 (dd, 1H, J ) 4.7, 13.1 Hz), 2.17-1.56
(m, 7H), 1.45 (t, 1H, J ) 12.5 Hz), 0.88 (s, 9H), 0.02 (s, 6H);
13C NMR (67.8 MHz, CDCl3) δ 169.9, 137.6, 137.51, 137.47,
137.2, 129.3, 128.2, 128.1, 127.7, 127.6, 127.4, 97.0, 95.0, 94.9,
93.5, 78.2, 77.1, 76.3, 72.3, 70.6, 69.9, 69.6, 69.3, 69.1, 67.9,
67.7, 53.7, 53.3, 53.2, 52.4, 52.3, 52.2, 52.1, 38.5, 25.7, 23.5,
18.00, -5.53; 31P NMR (CDCl3) δ 35.70; HR-ESI-MS calcd for
C53H77NO14PSi [M + H]+ 1010.4851, found 1010.4847.
Experimental Section
Methyl 5-Acetamido-2,6-anhydro-4,7,8,9-tetra-O-ben-
zyloxymethyl-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-
gluco-nononate (10). To a solution of methyl 5-acetamido-
2,6-anhydro-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-gluco-
nononate (9) (0.204 g, 0.59 mmol) in DMF (6.0 mL) were added
DIEA (0.83 mL, 4.7 mmol) and BOMCl (0.73 mL, 4.7 mmol).
After being stirred for 3 h at 60 °C, the solution was cooled
and diluted with EtOAc (100 mL). The organic layer was
washed with brine (50 mL × 3), dried (MgSO4), and concen-
trated. The residue was purified on a column of silica gel
(toluene-acetone 9:1) to yield 10 (452 mg, 93%) as a colorless
syrup: 1H NMR (270 MHz, CDCl3) δ 7.41-7.27 (m, 20H), 5.79
(d, 1H, J ) 7.3 Hz), 5.67-5.56 (m, 1H), 5.16-4.51 (m, 20H),
4.22 (d, 1H, J ) 9.9 Hz), 4.06 (m, 2H), 3.84 (dd, 1H, J ) 4.1,
9.9 Hz), 3.67 (s, 3H), 3.27 (dt, 1H, J ) 6.9, 10.2 Hz), 2.75 (m,
2H), 2.48 (dd, 1H, J ) 5.0, 13.2 Hz), 1.77 (dd, 1H, J ) 10.9,
13.2 Hz), 1.61 (s, 3H); 13C NMR (67.8 MHz, CDCl3) δ 172.0,
170.3, 137.8, 137.7, 137.6, 131.5, 128.5, 128.3, 128.0, 127.9,
127.6, 127.5, 127.4, 118.9, 96.7, 95.7, 95.1, 93.5, 79.3, 78.7, 77.4,
70.6, 69.9, 69.6, 69.4, 69.1, 68.5, 54.4, 52.0, 37.0, 36.7, 23.3;
HR-ESI-MS calcd for C47H58NO12 [M + H]+ 828.3959, found
828.3919.
5-Acetamido-2,6-anhydro-4,7,8,9-tetra-O-benzyloxy-
methyl-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-gluco-
nonitol (11). To a solution of 10 (401 mg, 0.48 mmol) in THF
(2.8 mL) was added LiEt3BH (1.0 M THF solution, 1.9 mL,
1.9 mmol) dropwise under Ar atmosphere. After being stirred
for 30 min at room temperature, the solution was diluted with
EtOAc (40 mL), and washed with water (10 mL). The organic
layer was dried (MgSO4) and concentrated. The residue was
purified on a column of silica gel (toluene-acetone 5:1) to yield
11 (379 mg, 98%) as a colorless syrup: 1H NMR (270 MHz,
CDCl3) δ 7.41-7.24 (m, 20H), 5.73-5.64 (m, 2H), 5.16-4.50
(m, 19H), 4.40 (d, 1H, J ) 10.6 Hz), 4.06 (d, 1H, J ) 10.2 Hz),
3.98 (m, 2H), 3.78 (dd, 1H, J ) 3.2, 10.6 Hz), 3.50-3.28 (m,
2H), 3.35 (dt, 1H, J ) 7.6, 10.2 Hz), 2.61 (dd, 1H, J ) 7.9, 13.9
Hz), 2.50 (br, 1H), 2.33 (dd, 1H, J ) 6.9, 13.9 Hz), 2.03 (dd,
1H, J ) 4.9, 12.9 Hz), 1.67 (s, 3H), 1.56 (t, 1H, J ) 12.6 Hz);
13C NMR (67.8 MHz, CDCl3) δ 170.2, 137.6, 137.50, 137.47,
137.0, 132.5, 129.9, 128.5, 128.33, 128.30, 128.28, 128.0,
127.61, 127.59, 127.58, 127.55, 127.5, 118.8, 96.8, 95.2, 94.7,
93.7, 77.6, 77.3, 70.9, 70.8, 69.7, 69.6, 69.4, 69.2, 68.5, 68.0,
54.5, 35.6, 34.7, 29.8, 23.7; HR-ESI-MS calcd for C46H58NO11
[M + H]+ 800.4010, found 800.3993.
5-Acetamido-2,6-anhydro-4,7,8,9-tetra-O-benzyloxy-
methyl-1-O-tert-butyldimethylsilyl-3,5-dideoxy-2-C-(2-
propenyl)-D-erythro-L-gluco-nonitol (12). To a solution of
11 (1.265 g, 1.58 mmol) in DMF (5.2 mL) was added imidazole
(0.742 g, 10.6 mmol). A solution of TBDMSCl (0.738 g, 4.92
mmol) in DMF (2.9 mL) was added to the solution at 0 °C.
After being stirred overnight at room temperature, the solution
was concentrated, and the residue was purified on a column
of silica gel (toluene-acetone 7:1) to yield 12 (1.345 g, 93%)
as a colorless syrup: 1H NMR (270 MHz, CDCl3) δ 7.40-7.26
(m, 20H), 5.76-5.69 (m, 2H), 5.17-4.50 (m, 19H), 4.37 (d, 1H,
J ) 10.2 Hz), 4.06 (d, 1H, J ) 9.9 Hz), 3.98 (m, 2H), 3.79 (dd,
1H, J ) 3.6, 9.9 Hz), 3.56 (d, 1H, J ) 9.9 Hz), 3.37 (d, 1H, J
) 9.9 Hz), 3.30 (m, 1H), 2.54-2.51 (m, 2H), 2.20 (dd, 1H, J )
5.0, 12.9 Hz), 1.64 (s, 3H), 1.37 (t, 1H, J ) 12.2 Hz), 0.87 (s,
Dimethyl 2-(5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-an-
hydro-1-O-tert-butyldimethylsilyl-3,5-dideoxy-D-erythro-
J. Org. Chem, Vol. 70, No. 22, 2005 8821

Izumi et al.
L-gluco-nonitol-2-yl)ethylphosphonate (15). To a solution
of 14 (0.256 g, 0.253 mmol) in MeOH (15 mL) were added Pd/C
(10%, 0.5 g) and HCO2NH4 (0.3 g, 7.9 mmol). After being
stirred for 2 h under reflux, another portion of HCO2NH4 (0.3
g, 7.9 mmol) was added. The suspension was stirred under
reflux for 3 h and filtered through a Celite pad, and the filtrate
was concentrated. The residue was acetylated with Ac2O (2
mL), pyridine (4 mL), and DMAP (cat.) and purified on a
column of silica gel (toluene-acetone 1:1) to yield 15 (0.121 g,
87%) as a colorless syrup: 1H NMR (270 MHz, CDCl3) δ 5.50
(d, 1H, J ) 9.9 Hz), 5.31 (dd, 1H, J ) 2.0, 5.9 Hz), 5.21 (dt,
1H, J ) 2.3, 6.3 Hz), 5.19-5.11 (m, 1H), 4.51 (dd, 1H, J ) 2.3,
12.5 Hz), 4.02 (dd, 1H, J ) 6.9, 12.5 Hz), 3.93 (q, 1H, J ) 10.2
Hz), 3.81 (dd, 1H, J ) 2.3, 9.2 Hz), 3.76, 3.75 (each d, 3H×2,
J ) 10.9 Hz), 3.53 (d, 1H, J ) 10.1 Hz), 3.38 (d, 1H, J ) 10.1
Hz), 2.17-1.61 (m, 6H), 2.12, 2.10, 2.02, 1.89 (each s, 3H, 3H,
6H, 3H), 0.89 (s, 9H), 0.07, 0.06 (each s, 3H×2); 13C NMR (67.8
MHz, CDCl3) δ 170.8, 170.4, 170.3, 170.1, 169.9, 77.3, 70.9,
70.8, 69.9, 68.4, 67.6, 62.4, 52.6, 52.51, 52.48, 52.38, 50.2, 37.0,
25.8, 23.2, 22.55, 22.51, 21.07, 21.05, 20.8, 18.9, 18.1, 16.8,
-5.45, -5.50; 31P NMR (CDCl3) δ 35.56; HR-ESI-MS calcd for
C29H52NO14PSiNa [M + Na]+ 720.2787, found 720.2791.
Dimethyl 2-(5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-an-
hydro-3,5-dideoxy-D-erythro-L-gluco-nonitol-2-yl)ethyl-
phosphonate (16). To a solution of 15 (0.121 g, 0.174 mmol)
in THF (8.2 mL) was added HF-pyridine (70% HF, 0.5 mL)
at 0 °C. The solution was stirred overnight at room temper-
ature, and the reaction was quenched by adding saturated
aqueous NaHCO3. The aqueous layer was extracted twice with
CHCl3, and the combined organic layer was dried (MgSO4) and
concentrated. The residue was purified on a column of silica
gel (EtOAc-MeOH 10:1f9:1) to yield 16 (81.6 mg, 80%) as a
syrup: 1H NMR (270 MHz, CDCl3) δ 5.71 (d, 1H, J ) 9.2 Hz),
5.32 (dd, 1H, J ) 2.1, 5.8 Hz), 5.25-5.10 (m, 2H), 4.59 (dd,
1H, J ) 2.3, 12.5 Hz), 4.13-3.86 (m, 3H), 3.77, 3.76 (each d,
3H×2, J ) 10.9 Hz), 3.54-3.42 (m, 2H), 2.87 (br, 1H), 2.19-
1.62 (m, 6H), 2.14, 2.11, 2.04, 2.01, 1.89 (each s, 3H×5); 13C
NMR (67.8 MHz, CDCl3) δ 170.7, 170.5, 170.3, 170.1, 169.8,
77.7, 71.0, 69.8, 68.6, 67.3, 62.6, 52.5, 52.4, 49.6, 34.6, 23.0,
22.2, 20.9, 20.7, 19.1, 17.0; 31P NMR (CDCl3) δ 34.96; HR-ESI-
MS calcd for C23H38NO14PNa [M + Na]+ 606.1928, found
606.1923.
mmol) in Et2O-EtOH (2:1 v/v, 8.1 mL) and heating at 70 °C.
The solution stood for 3 h. Another batch of CH2N2 solution
was added to the solution since TLC showed remaining 17.
After standing for 1 h, the solution was still yellow and TLC
showed the disappearance of 17. AcOH was added to the
solution until the yellow color disappeared. The solution was
concentrated, and remaining AcOH was coevaporated with
toluene. The residue was purified on a column of silica gel
(EtOAc-MeOH 10:1) to yield 18 (85.5 mg, 81%) as a syrup:
1H NMR (270 MHz, CDCl3) δ 5.79 (d, 1H, J ) 8.2 Hz), 5.38 (d,
1H, J ) 3.3 Hz), 5.24-5.20 (m, 2H), 4.81 (dd, 1H, J ) 2.3,
12.5 Hz), 4.11 (dd, 1H, J ) 7.9, 12.5 Hz), 3.99-3.83 (m, 2H),
3.77 (s, 3H), 3.77 (d, 3H, J ) 10.6 Hz), 3.76 (d, 3H, J ) 10.9
Hz), 2.30 (dd, 1H, J ) 5.1, 13.6 Hz), 2.38-1.56 (m, 5H), 2.15,
2.08, 2.03, 2.02, 1.89 (each s, 3H×5); 13C NMR (67.8 MHz,
CDCl3) δ 171.0, 170.55, 170.48, 170.4, 170.1, 170.0, 79.1, 78.8,
72.0, 71.6, 69.1, 68.7, 68.8, 62.6, 52.8, 52.7, 52.6, 52.5, 49.4,
36.4, 24.8, 23.2, 21.0, 20.9, 20.84, 20.80, 19.4, 17.3; 31P NMR
(CDCl3) δ 34.19; HR-ESI-MS calcd for C24H39NO15P [M + H]+
612.2057, found 612.2096.
Triethylammonium Methyl 2-(Methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-erythro-
L-gluco-nonanoate-2-yl)ethylphosphonate (19). To a so-
lution of 18 (0.114 g, 0.187 mmol) in 1,4-dioxane (0.95 mL)
were added PhSH (0.74 mL) and Et3N (1.5 mL). After the
reaction stirred for 4 h at room temperature, MeOH was added
to the solution. The solution was concentrated, and the residue
was purified on a column of silica gel (EtOAc-MeOH-Et3N
4:1:0.1f3:1:0.1) to yield 19 (0.116 g, 87%) as a syrup: 1H NMR
(270 MHz, CDCl3) δ 6.70 (br, 1H), 5.40 (t, 1H, J ) 2.3 Hz),
5.24-5.17 (m, 2H), 4.90 (dd, 1H, J ) 2.0, 12.5 Hz), 4.21 (dd,
1H, J ) 8.2, 12.5 Hz), 4.16 (overlap, 1H), 4.03 (q, 1H, J ) 10.2
Hz), 3.73 (s, 3H), 3.57 (d, 3H, J ) 10.6 Hz), 3.04 (q, 5H, J )
7.3 Hz), 2.36 (dd, 1H, J ) 4.6, 12.9 Hz), 2.51-2.33, 2.08-1.81,
1.58-1.37 (each m, 1H, 3H, 1H), 2.14, 2.06, 2.02, 1.99, 1.88
(each s, 3H×5), 1.31 (t, 9H, J ) 7.3 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 171.6, 170.24, 170.19, 170.10, 170.08, 169.9, 79.3,
79.1, 72.3, 71.1, 69.7, 68.9, 62.7, 52.2, 51.4, 51.3, 48.7, 45.1,
36.2, 26.1, 23.0, 20.9, 20.8, 20.7, 18.7, 8.4; 31P NMR (CDCl3) δ
24.96; HR-ESI-MS calcd for C23H37NO15P [M - Et3N + H]+
598.1901, found 598.1904.
Sodium Cytidin-5′-yl 2-(Sodium 5-acetamido-2,6-an-
hydro-3,5-dideoxy-D-erythro-L-gluco-nonanoate-2-yl)eth-
ylphosphonate (4) and Sodium Cytidin-5′-yl 2-(5-Aceta-
mido-2,6-anhydro-3,5-dideoxy-D-erythro-L-gluco-nona-
namide-2-yl)ethylphosphonate (5). Compound 19 and 4-N-
acetyl-2′,3′-di-O-acetylcytidine (20) were dried by concentrating
three times from dry DMF in vacuo prior to use. To a solution
of 19 (0.112 g, 0.158 mmol) and 20 (0.175 g, 0.474 mmol) in
DMF (0.87 mL) were added BOP (0.210 g, 0.475 mmol) and
DIEA (87 µL, 0.63 mmol). After being stirred for 3 h at room
temperature, the solution was diluted with EtOAc, and washed
twice with brine. The organic layer was dried (MgSO4) and
concentrated. The residue was purified on a column of silica
gel (EtOAc-MeOH 10:1f7:1), first to yield unreacted 20, and
next 4-N-acetyl-2′,3′-di-O-acetyl-cytidin-5′-yl methyl 2-(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-
erythro-L-gluco-nonanoate-2-yl)ethylphosphonate (0.169 g,
HMPA contamination, <90%) as a syrup in a diastereomer
mixture on P: 31P NMR (CDCl3) δ 33.83, 33.27; HR-ESI-MS
calcd for C38H54N4O22P [M + H]+ 949.2967, found 949.2959.
To a solution of the syrup (0.169 g, HMPA, contamination,
<0.142 mmol) in 1,4-dioxane (0.72 mL) were added PhSH (0.56
mL) and Et3N (1.1 mL). After being stirred overnight at room
temperature, MeOH was added to the solution. The solution
was concentrated, and the residue was purified on a column
of silica gel (EtOAc-MeOH 2% Et3N 4:1f3:1) to yield triethyl-
ammonium 4-N-acetyl-2′,3′-di-O-acetylcytidin-5′-yl 2-(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-
erythro-L-gluco-nonanoate-2-yl)ethylphosphonate (21, 0.130 g,
<81%) as a syrup: 1H NMR (270 MHz, CDCl3) δ 8.45 (d, 1H,
J ) 7.6 Hz), 7.82, 7.63 (each d, 1 H, J ) 6.6 Hz), 7.39 (d, 1H,
Dimethyl 2-(5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-an-
hydro-3,5-dideoxy-D-erythro-L-gluco-nonanoic acid-2-yl)-
ethylphosphonate (17). To a solution of 16 (0.121 g, 0.208
mmol) in CCl4-MeCN-H2O (2:2:3 v/v/v, 1.5 mL) was added
NaIO4 (0.182 g, 0.851 mmol) and the mixture stirred until all
solids dissolved. RuCl3‚nH2O (1.2 mg, 6 µmol) was added to
the solution and the mixture stirred for 3 h at room temper-
ature. Precipitate was formed during the reaction. The sus-
pension was diluted with CHCl3 and washed with saturated
aqueous NaHCO3. The aqueous layer was extracted three
times with CHCl3. The combined organic layer was dried
(MgSO4) and concentrated. The residue was purified on a
column of silica gel (CHCl3-MeOH 4:1f3:1) to yield 17 (0.116
g, 93%) as a syrup: 1H NMR (270 MHz, CDCl3-CD3OD 2:1) δ
5.37 (dd, 1H, J ) 1.3, 4.6 Hz), 5.32-5.30 (m, 1H), 5.22 (dt,
1H, J ) 4.9, 10.4 Hz), 4.78 (dd, 1H, J ) 1.6, 12.2 Hz), 4.14
(dd, 1H, J ) 6.9, 12.5 Hz), 3.97-3.83 (m, 2H), 3.79, 3.77 (each
d, 3H×2, J ) 10.9 Hz), 2.31 (dd, 1H, J ) 4.9, 12.5 Hz), 2.14-
1.72 (m, 5H), 2.14, 2.09, 2.03, 2.02, 1.89 (each s, 3H×5); 13C
NMR (67.8 MHz, CDCl3-CD3OD 2:1) δ 171.2, 170.7, 170.4,
169.8, 78.6, 71.0, 70.6, 69.4, 68.2, 62.2, 52.3, 52.2, 52.1, 49.15,
49.05, 36.3, 23.7, 22.0, 20.3, 20.2, 20.1, 20.0, 18.9, 17.3, 16.8;
31P NMR (CDCl3-CD3OD 2:1) δ 39.83; HR-ESI-MS calcd for
C23H37NO15P [M + H]+ 598.1901, found 598.1921.
Dimethyl 2-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
2,6-anhydro-3,5-dideoxy-D-erythro-L-gluco-nonanoate-2-
yl)ethylphosphonate (18). To a solution of 17 (0.104 g, 0.173
mmol) in benzene-EtOH (4:1 v/v, 8.0 mL) was added CH2N2
in Et2O by distillation, which was generated by adding 50%
KOH (0.123 mL) to a solution of TsN(NO)Me (0.161 g, 0.752
8822 J. Org. Chem., Vol. 70, No. 22, 2005

Bisubstrate and Donor Analogues of Sialyltransferase
J ) 7.3 Hz), 6.94 (br, 1H), 6.24 (d, 1H, J ) 5.3 Hz), 5.61-5.48
(m, 3H), 5.17-5.10 (m, 2H), 4.87 (d, 1H, J ) 10.6 Hz), 4.38-
4.07 (m, 6H), 3.73 (s, 3H), 2.99 (q, 6H, J ) 7.3 Hz), 2.37-1.86
(m, 6H), 2.23, 2.14, 2.11, 2.07, 2.02, 1.982, 1.976, 1.86 (each s,
3H×8), 1.25 (9H, t, J ) 7.3 Hz); 13C NMR (67.8 MHz, CDCl3)
δ 171.5, 171.2, 170.5, 170.3, 170.2, 169.9, 169.8, 162.9, 155.5,
145.6, 97.4, 87.6, 82.2, 82.0, 79.7, 79.5, 74.4, 72.6, 71.7, 71.4,
70.0, 69.3, 63.0, 62.9, 52.4, 48.8, 45.4, 35.7, 26.89, 26.85, 24.9,
23.1, 22.50, 22.45, 21.0, 20.9, 20.7, 20.6, 8.8; 31P NMR (CDCl3)
δ 22.94; HR-ESI-MS calcd for C37H52N4O22P [M + H]+ 935.2811,
found 935.2821.
g, 0.328 mmol) in DMF (0.6 mL) was treated with DIEA (60
µL, 0.433 mmol) and BOP (0.147 g, 0.332 mmol) as mentioned
for the synthesis of 21. Purification by silica gel column
chromatography (EtOAc-MeOH 20:1f15:1f10:1f9:1f8:
1f7:1) yielded 4-N-acetyl-2′,3′-di-O-acetylcytidin-5′-yl methyl
2-(5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-1-O-tert-bu-
tyldimethylsilyl-3,5-dideoxy-D-erythro-L-gluco-nonitol-2-yl)eth-
ylphosphonate (23, 0.115 g, HMPA contamination, <89%) as
a syrup, which was a diastereomer mixture on P: 1H NMR
(270 MHz, CDCl3) δ 9.24 (brs, 1H), 7.70-7.11 (m, 3H), 5.87-
5.70 (m, 3H), 5.45 (d, 1H, J ) 2.6 Hz), 5.11-5.22 (m, 2H),
4.58-3.92 (m, 7H), 3.75-3.68 (m, 3H), 3.54-3.33 (m, 2H),
2.17-1.60 (m, 30H), 0.886, 0.880 (each s, 9H), 0.08, 0.06 (each
s, 6H); 31P NMR (CDCl3) δ 35.45, 35.33; HR-ESI-MS calcd for
C43H68N4O21PSi [M + H]+ 1035.3883, found 1035.3898.
A solution of 23 (23.9 mg, HMPA contamination, <20 µmol)
in 1,4-dioxane (0.1 mL) was treated with PhSH (78 µL) and
Et3N (0.16 mL) as mentioned for 18. Purification by silica gel
column chromatography (EtOAc-MeOH 6:1fEtOAc-MeOH-
Et3N 4:1:0.2f3:1:0.2f5:2:0.4) yielded triethylammonium 4-N-
acetyl-2′,3′-di-O-acetylcytidin-5′-yl 2-(5-acetamido-4,7,8,9-tetra-
O-acetyl-2,6-anhydro-1-O-tert-butylsimethylsilyl-3,5-dideoxy-
D-erythro-L-gluco-nonitol-2-yl)ethylphosphonate (23.8 mg, HMPA
contamination, <20 µmol, <99%) as a syrup: 31P NMR (CDCl3)
δ 26.28; HR-ESI-MS calcd for C42H66N4O21PSi [M + H]+
1021.3727, found 1021.3812.
A solution of the syrup (23.8 mg, HMPA contamination, <20
µmol) in THF-HCO2H-H2O (6:3:1 v/v/v, 2.0 mL) was stirred
for 5 h at room temperature. MeOH was added to the solution,
and the solution was concentrated in vacuo. The residue was
purified on a column of spherical silica gel (EtOAc-MeOH-
Et3N 4:1:0.2f3:1:0.1f2:1:0.1) to yield triethylammonium 4-N-
acetyl-2′,3′-di-O-acetylcytidin-5′-yl 2-(5-acetamido-4,7,8,9-tetra-
O-acetyl-2,6-anhydro-3,5-dideoxy-D-erythro-L-gluco-nonitol-2-
yl)ethylphosphonate (21 mg, small HMPA contamination, 99%)
as a syrup: 31P NMR (CDCl3) δ 23.85; HR-ESI-MS calcd for
C36H52N4O21P [M + H]+ 907.2862, found 907.2867.
A solution of the syrup (21 mg, <20 µmol) in MeOH (2 mL)
and NH4OH (28%, 2 mL) was treated as mentioned for the
synthesis of 4. Purification by a DEAE-Sephadex A-25 column
(0-1 M HCO2Na linear gradient) and Sephadex G-15 chro-
matography (water) as mentioned for 4 yielded 6 (8.3 mg, 63%)
as a white solid after lyophilization: 1H NMR (400 MHz, D2O,
COSY) δ 7.96 (d, 1H, J ) 7.6 Hz, H6), 6.13 (d, 1H, J ) 7.6 Hz,
H5), 6.03 (d, 1H, J ) 3.8 Hz, H1′), 4.37-4.33 (m, 2H, H2′,3′),
4.29 (br, 1H, H4′), 4.14 (m, 1H, H5′a), 4.08 (m, 1H, H5′b), 3.89
(m, 1H, H4′′), 3.84-3.79 (m, 2H, H8′′,9′′a), 3.76 (t, 1H, J )
10.2 Hz, H5′′), 3.66 (d, 1H, J ) 10.2 Hz, H6′′), 3.66 (dd, 1H, J
) 6.6, 12.6 Hz, H9′′b), 3.60 (d, 1H, J ) 12.0 Hz, H1′′a), 3.50
(d, 1H, J ) 9.0 Hz, H7′′), 3.45 (d, 1H, J ) 12.0 Hz, H1′′b), 2.06
(s, 3H, Ac), 2.05-1.96 (m, 1H, CH2CH2), 1.92 (dd, 1H, J ) 4.6,
12.5 Hz, H3′′eq), 1.72 (t, 1H, J ) 12.5 Hz, H3′′ax), 1.81-1.53
(m, 3H, CH2CH2); 13C NMR (100 MHz, D2O) δ 175.9, 167.2,
158.8, 142.5, 97.4, 90.4, 83.91, 83.82, 77.63, 77.47, 75.0, 70.9,
70.8, 70.5, 69.6, 68.6, 67.0, 64.5, 63.92, 63.87, 53.7, 37.9, 25.2,
23.1, 20.6, 19.3; 31P NMR (D2O) δ 28.84; HR-ESI-MS calcd for
C22H38N4O14P [M + H]+ 613.2122, found 613.2121.
To a solution of the syrup (0.129 g, 0.114 mmol) in MeOH
(5 mL) was added 28% NH4OH (5 mL) and the mixture stirred
for 6 h at room temperature. The solution was concentrated
in vacuo, and the residue was applied to a DEAE-Sephadex
A-25 column. The column was first washed with water and
then eluted with a 0-1 M linear gradient of HCO2Na solution.
Two peaks were detected by UV absorbance at 275 nm around
0.1 and 0.3 M. Fractions containing each peak were collected,
separately, and lyophilized. Each residue was purified by
Sephadex G-15 chromatography (water) and lyophilized to
yield 5 (21.9 mg, 30%), which was the first peak, and 4 (24.5
mg, 32%), which was the second peak, as white solids.
1
Compound 4: H NMR (400 MHz, D2O, COSY) δ 8.09 (d, 1H,
J ) 7.6 Hz, H6), 6.24 (d, 1H, J ) 7.6 Hz, H5), 6.01 (d, 1H, J
) 3.4 Hz, H1′), 4.37-4.33 (m, 2H, H2′,3′), 4.30 (br, 1H, H4′),
4.17 (m, 1H, H5′a), 4.07 (m, 1H, H5′b), 3.97-3.84 (m, 3H,
H4′′,8′′,9′′a), 3.81 (t, 1H, J ) 10.1 Hz, H5′′), 3.66 (d, 1H, J )
9.0 Hz, H6′′), 3.66 (dd, 1H, J ) 5.5, 11.9 Hz, H9′′b), 3.48 (d,
1H, J ) 9.3 Hz, H7′′), 2.27 (dd, 1H, J ) 4.6, 13.1 Hz, H3′′eq),
2.06 (s, 3H, Ac), 2.22-1.91 (m, 2H, CH2P), 1.68 (t, 1H, J )
12.3 Hz, H3′′ax), 1.84-1.73, 1.44-1.33 (each m, 1H×2, CH2C);
13C NMR (100 MHz, D2O) δ 180.7, 175.6, 166.3, 157.7, 142.7,
97.3, 90.2, 84.06, 83.98, 81.41, 81.21, 75.0, 71.0, 70.9, 70.3, 69.7,
68.8, 64.5, 63.52, 63.47, 53.6, 40.9, 26.5, 23.1, 21.8, 20.5; 31P
NMR (D2O) δ 28.47; HR-ESI-MS calcd for C22H36N4O15P [M +
H]+ 627.1909, found 627.1924. Compound 5: 1H NMR (400
MHz, D2O, COSY) δ 7.98 (d, 1H, J ) 7.5 Hz, H6), 6.17 (brd,
1H, J ) 6.4 Hz, H5), 6.03 (d, 1H, J ) 3.8 Hz, H1′), 4.37-4.30
(m, 2H, H2′,3′), 4.27 (br, 1H, H4′), 4.13 (m, 1H, H5′a), 4.05
(m, 1H, H5′b), 3.94-3.82 (m, 4H, H4′′,5′′,8′′,9′′a), 3.74 (d, 1H,
J ) 9.8 Hz, H6′′), 3.67 (dd, 1H, J ) 5.3, 11.9 Hz, H9′′b), 3.58
(d, 1H, J ) 9.6 Hz, H7′′), 2.25 (dd, 1H, J ) 4.0, 13.2 Hz, H3′′eq),
2.06 (s, 3H, Ac), 2.20-1.92 (m, 2H, CH2P), 1.70 (t, 1H, J )
11.7 Hz, H3′′ax), 1.89-1.77, 1.43-1.31 (each m, 1H×2, CH2C);
13C NMR (100 MHz, D2O) δ 178.9, 175.9, 166.6, 158.1, 142.7,
97.5, 90.3, 83.98, 83.90, 80.64, 80.47, 75.0, 71.5, 70.6, 70.5, 69.1,
68.3, 64.4, 63.90. 63.85, 53.4, 40.3, 25.9, 23.1, 21.0, 19.6; 31P
NMR (D2O) δ 27.56; HR-ESI-MS calcd for C22H37N5O15P [M +
H]+ 626.2069, found 626.2077.
Triethylammonium Methyl 2-(5-Acetamido-4,7,8,9-
tetra-O-acetyl-2,6-anhydro-1-O-tert-butyldimethylsilyl-
3,5-dideoxy-D-erythro-L-gluco-nonitol-2-yl)ethylphospho-
nate (22). A solution of 15 (0.214 g, 0.306 mmol) in 1,4-dioxane
(1.6 mL) was treated with PhSH (1.2 mL) and Et3N (2.5 mL),
as mentioned for 18. Purification by silica gel column chro-
matography (EtOAc-MeOH-Et3N 4:1:0.1f3:1:0.1) yielded 22
(0.208 g, 86%) as a syrup: 1H NMR (270 MHz, CDCl3) δ 7.15
(d, 1H, J ) 9.9 Hz), 5.34 (dd, 1H, J ) 2.0, 5.0 Hz), 5.27-5.13
(m, 2H), 4.55 (dd, 1H, J ) 2.3, 12.2 Hz), 4.13-3.92 (m, 3H),
3.62 (d, 3H, J ) 10.6 Hz), 3.52 (d, 1H, J ) 9.9 Hz), 3.39 (d,
1H, J ) 9.9 Hz), 3.09 (q, 6H, J ) 7.3 Hz), 2.12-1.87 (m, 5H),
2.12, 2.10, 2.01, 1.99, 1.87 (each s, 3H×5), 1.64 (t, 1H, J )
12.2 Hz), 1.33 (t, 9H, J ) 7.3 Hz), 0.88 (s, 9H), 0.06, 0.05 (each
s, 3H×2); 13C NMR (67.8 MHz, CDCl3) δ 170.4, 170.3, 170.2,
169.9, 77.2, 70.9, 70.3, 68.6, 67.6, 62.5, 51.64, 51.56, 49.3, 45.4,
36.3, 25.8, 23.4, 23.0, 21.1, 20.8, 20.7, 20.0, 18.1, 18.0, 8.5, -5.4,
-5.5; 31P NMR (CDCl3) δ 28.42; HR-ESI-MS calcd for C28H51-
NO14PSi [M - NEt3 + H]+ 684.2816, found 684.2846.
Sodium Cytidin-5′-yl 2-{5-Acetamido-2,6-anhydro-1-
(sodium ethyl phosphate)-3,5-dideoxy-D-erythro-L-gluco-
nonitol-2-yl}ethylphosphonate (7). A solution of 23 (0.228
g, HMPA contamination, <0.145 mmol) in TBAF in THF (1
M, 1.0 mL, pH adjusted to 4-5 with AcOH) was stirred at
room temperature for 22 h. The solution was concentrated,
and the residue was purified on a column of silica gel (EtOAc-
MeOH 15:1f10:1f9:1) to yield 4-N-acetyl-2′,3′-di-O-acetylcy-
tidin-5′-yl methyl 2-(5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-
anhydro-3,5-dideoxy-D-erythro-L-gluco-nonitol-2-yl)ethylphos-
phonate (24, 0.129 g, HMPA contamination, <64%) as a syrup,
which was a diastereomer mixture on P: 1H NMR (270 MHz,
CDCl3) δ 9.25 (brs, 1H), 7.73-7.20 (m, 3H), 5.86-5.69 (m, 3H),
5.47, 5.36 (each d, 1H, J ) 2.6 Hz), 5.21-5.11 (m, 2H), 4.70-
4.03 (m, 6H), 3.90-3.68 (m, 6H), 2.31-1.57 (m, 30H); 31P NMR
Sodium Cytidin-5′-yl 2-(5-Acetamido-2,6-anhydro-3,5-
dideoxy-D-erythro-L-gluco-nonitol-2-yl)ethylphosphon-
ate (6). A solution of 22 (85.9 mg, 0.109 mmol) and 20 (0.121
J. Org. Chem, Vol. 70, No. 22, 2005 8823

Izumi et al.
(CDCl3) δ 34.47, 34.08; HR-ESI-MS calcd for C37H54N4O21P [M
+ H]+ 921.3018, found 921.3007.
4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-1-{triethylam-
monium (methyl 2,3,4-tri-O-acetyl-â-D-galactopyranosid-6-yl)
phosphate}-D-erythro-L-gluco-nonitol-2-yl]ethylphosphonate (26,
16.9 mg, 43%) as a syrup: 1H NMR (270 MHz, CDCl3) δ 8.40
(brs, 1H), 7.30 (overlap), 6.88 (brs, 1H), 6.15 (brs, 1H), 5.57-
4.95 (m, 10H), 4,52-3.50 (m, 12H), 3.50 (s, 3H), 3.05 (q, J )
7.3 Hz), 2.25-1.53 (m, 39H), 1.29 (t, J ) 7.3 Hz); 31P NMR
(CDCl3) δ 24.33, -0.44; m/z (ESI) 1289.3 (M - 2Et3N + H+).
To a solution of 26 (16.9 mg) in MeOH (2 mL) was added
NH4OH (28%, 2 mL), and the mixture stirred overnight at
room temperature. The solution was concentrated in vacuo.
Purification of the residue by DEAE-Sephadex A-25 column
(0-1 M HCO2NH4 linear gradient) and Sephadex G-15 chro-
matography (water) as mentioned for 4 yielded 8 (5.4 mg, 53%)
as a white solid: 1H NMR (400 MHz, D2O, COSY) δ 8.04 (d,
1H, J ) 7.6 Hz, H6), 6.20 (brd, 1H, J ) 6.4 Hz, H5), 6.03 (d,
1H, J ) 3.7 Hz, H1′), 4.36-4.33 (m, 3H, H2′,3′,1′′′), 4.30 (br,
1H, H4′), 4.16 (m, 1H, H5′a), 4.09 (m, 1H, H5′b), 4.03 (t, 2H,
J ) 6.6 Hz, H6′′′a,b), 3.99 (d, 1H, J ) 3.5 Hz, H4′′′), 3.91 (dt,
1H, J ) 4.6, 11.0 Hz, H4′′), 3.86-3.76 (m, 6H, H1′′a,b,5′′,8′′,9′′a,-
5′′′), 3.69 (d, 1H, J ) 9.3 Hz, H6′′), 3.67 (dd, 1H, J ) 3.4, 10.1
Hz, H3′′′), 3.62 (dd, 1H, J ) 5.8, 11.8 Hz, H9′′b), 3.58 (s, 3H,
OMe), 3.52 (dd, 1H, J ) 7.9, 9.9 Hz, H2′′′), 3.47 (d, 1H, J )
9.6 Hz, H7′′), 2.12-2.03 (m, 2H, H3′′eq, CH2CH2), 2.05 (s, 3H,
Ac), 1.78-1.74 (m, 3H, CH2CH2), 1.66 (t, 1H, J ) 12.2 Hz,
H3′′ax); 13C NMR (100 MHz, D2O) δ 175.8, 166.0, 157.3, 142.8,
104.8, 97.5, 90.2, 84.07, 84.00, 76.89, 76.80, 76.72, 76.63, 75.1,
74.57, 74.49, 73.6, 71.7, 71.0, 70.8, 70.4, 69.6, 69.2, 68.5, 65.08,
65.03, 64.4, 63.65, 63.61, 58.2, 53.7, 39.0, 24.9, 23.1, 20.7, 19.4;
31P NMR (D2O) δ 28.75, 1.04; HR-ESI-MS calcd for C29H51N4-
O22P2 [M - 2NH3 + H]+ 869.2470, found 869.2461.
R-2,3-ST Assay. The reaction mixture containing 0.11 mM
CMP-Neu5Ac, 1.0 mM PA-LacNAc, 1 mg/mL BSA, 0.5%
Triton X-100, 350 µU R-2,3-ST, and inhibitor in 50 mM HEPES
buffer (pH 7.4) was adjusted to total volume of 20 µL. The
reaction solution was incubated at 25 °C for 20 min. An aliquot
(10 µL) of the reaction mixture was added to 0.1 M AcOH (70
µL) to stop the reaction, and the amount of the reaction product
was analyzed by RP-HPLC (Inertsil-ODS-3 φ4.6 mm×100
mm). The column was heated at 40 °C and eluted with 0.1 M
NH4OAc (pH 4.5) containing 0.025% 1-BuOH at the flow rate
of 1.5 mL/min. The reaction product was quantitated with
fluorescence detector with excitation at 320 nm and emission
at 400 nm. Retention time of the reaction product was 11.09
min. In each assay, the amount of the product formed was less
than 10% of the amount of added CMP-Neu5Ac.
To a solution of 24 (44.2 mg, HMPA contamination, <31.9
µmol) in CH2Cl2 (0.5 mL) were added DIEA (18 µL) and diethyl
phosphorchloridite (14 µL) under Ar atmosphere. After being
stirred for 2 h at room temperature, the solution was concen-
trated. The residue was dissolved in MeCN (0.5 mL), and to
the solution was added tBuOOH (2.5 M decane solution, 80
µL). After being stirred for 30 min at room temperature, the
solution was concentrated, and the residue was purified on a
column of silica gel (EtOAc-MeOH 10:1) to yield 4-N-acetyl-
2′,3′-di-O-acetylcytidin-5′-yl methyl 2-{5-acetamido-4,7,8,9-
tetra-O-acetyl-2,6-anhydro-1-(diethyl phosphate)-3,5-dideoxy-
D-erythro-L-gluco-nonitol-2-yl}ethylphosphonate (39.5 mg, HMPA
contamination) as a syrup: HR-ESI-MS calcd for C41H63N4O24P2
[M + H]+ 1057.3308, found 1057.3291.
To a solution of the syrup (39.5 mg, HMPA contamination)
in MeCN (2 mL) was added LiBr (11 mg). After being stirred
under reflux overnight, the solution was concentrated to yield
crude lithium 4-N-acetyl-2′,3′-di-O-acetylcytidin-5′-yl 2-{5-
acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-1-
(lithium ethyl phosphate)-D-erythro-L-gluco-nonitol-2-yl}ethyl-
phosphonate as a syrup: m/z (ESI) 1015.0 (M - 2Li + 3H+).
To a solution of the crude syrup in MeOH (2 mL) was added
28% NH4OH (2 mL). After being stirred for 8 h at room
temperature, the solution was concentrated. Purification of the
residue by DEAE-Sephadex A-25 column (0-1 M HCO2Na
linear gradient) and Sephadex G-15 chromatography (water)
as mentioned for 4 yielded 7 (12.1 mg, 50% from 24) as a white
solid: 1H NMR (400 MHz, D2O, COSY) δ 8.00 (d, 1H, J ) 7.6
Hz, H6), 6.16 (d, 1H, J ) 7.5 Hz, H5), 6.04 (d, 1H, J ) 3.8 Hz,
H1′), 4.36-4.32 (m, 2H, H2′,3′), 4.30 (br, 1H, H4′), 4.16 (ddd,
1H, J ) 2.4, 3.8, 11.7 Hz, H5′a), 4.10 (ddd, 1H, J ) 2.4, 4.8,
11.7 Hz, H5′b), 3.94 (qu, 2H, J ) 7.2 Hz, Et), 3.91 (dt, 1H, J
) 5.0, 11.4 Hz, H4′′), 3.84-3.76 (m, 5H, H1′′a,b,5′′,8′′,9′′a), 3.69
(d, 1H, J ) 11.0 Hz, H6′′), 3.63 (dd, 1H, J ) 6.6, 12.6 Hz, H9′′b),
3.48 (d, 1H, J ) 8.9 Hz, H7′′), 2.08-2.03 (m, 2H, H3′′eq, CH2-
CH2), 2.06 (s, 3H, Ac), 1.80-1.62 (m, 3H, CH2CH2), 1.67 (t,
1H, J ) 12.1 Hz, H3′′ax), 1.27 (t, 3H, J ) 7.1 Hz, Me); 13C
NMR (100 MHz, D2O) δ 175.8, 167.1, 158.7, 142.3, 97.5, 90.1,
83.98, 83.89, 76.89, 76.80, 76.73, 76.63, 75.1, 71.0, 70.8, 70.5,
70.26, 70.21, 69.6, 68.5, 64.4, 63.68, 63.63, 63.41, 63.36, 53.7,
38.9, 24.9, 23.1, 20.8, 19.4, 16.69, 16.62; 31P NMR (D2O) δ
28.69, 1.21; HR-ESI-MS calcd for C24H43N4O17P2 [M - 2Na +
3H]+ 721.2099, found 721.2103.
Ammonium Cytidin-5′-yl 2-[5-Acetamido-2,6-anhydro-
3,5-dideoxy-1-{ammonium (methyl â-D-galactopyranosid-
6-yl) phosphate}-D-erythro-L-gluco-nonitol-2-yl]ethylphos-
phonate (8). Compound 24 and 2-cyanoethyl (methyl 2,3,4-
tri-O-acetyl-â-D-galactopyranosid-6-yl) N,N-diisopropylphos-
phoramidite (25) were dried by concentrating twice from dry
toluene then in vacuo prior to use. To a solution of 24 (29.1
mg, HMPA contamination, <26 µmol) and 25 (27.5 mg, 52.8
µmol) in dry MeCN (0.35 mL) was added 1H-tetrazole (5.5 mg,
78 µmol) under Ar atmosphere. After stirring for 50 min at
room temperature, tBuOOH (2.5 M decane solution, 30 µL,
75 µmol) was added. The solution was stirred for 30 min at
room temperature and then concentrated in vacuo to yield 4-N-
acetyl-2′,3′-di-O-acetylcytidin-5′-yl methyl 2-[5-acetamido-
4,7,8,9-tetra-O-acetyl-2,6-anhydro-1-{2-cyamoethyl (methyl 2,3,4-
tri-O-acetyl-â-D-galactopyranosid-6-yl) phosphate}-3,5-dideoxy-
D-erythro-L-gluco-nonitol-2-yl]ethylphosphonate as a syrup:
m/z (ESI) 1356.4 (M + H+).
R-2,6-ST Assay. The reaction mixture containing 0.11 mM
CMP-Neu5Ac, 1.0 mM PA-LacNAc, 1 mg/mL BSA, 0.5%
Triton X-100, 227 µU R-2,6-ST, and inhibitor in 25 mM sodium
cacodylate buffer (pH 6.8) was adjusted to a total volume 20
µL. The reaction solution was incubated at 25 °C for 15 min.
The reaction was stopped and the amount of the product was
quantitated as mentioned for the R-2,3-ST assay. The retention
time of the reaction product was 11.54 min. In each assay,
the amount of the product formed was less than 10% of the
amount of added CMP-Neu5Ac.
Acknowledgment. This research was partially sup-
ported by a Grant-in-Aid for Young Scientists (B) from
the Ministry of Education, Culture, Sports, Science and
Technology (14780457).
Supporting Information Available: General experimen-
tal procedures and 1H and 13C NMR spectra of compounds 4-8,
10, 12-19, 21-24, and 26. This material is available free of
charge via the Internet at http://pubs.acs.org.
A solution of the syrup in 1,4-dioxane (0.2 mL) was treated
with PhSH (0.1 mL) and Et3N (0.32 mL) as mentioned for 18.
Purification by silica gel column chromatography (EtOAc-
MeOH 2:1f1:1, containing 1% Et3N) yielded triethylammo-
nium 4-N-acetyl-2′,3′-di-O-acetylcytidin-5′-yl 2-[5-acetamido-
JO0512608
8824 J. Org. Chem., Vol. 70, No. 22, 2005