Synthesis and Characterization of an Anomeric Sulfur Analogue of CMP-Sialic Acid

Article abstract of DOI:10.1021/jo000646+

α-2,3-Sialyltransferase catalyzes the transfer of sialic acid from CMP-sialic acid (1) to a lactose acceptor. An analogue of 1 was synthesized in which the anomeric oxygen atom was replaced with a sulfur atom (1S). The key step in the synthesis of IS was a tetrazole-promoted coupling of a cytidine-5′-phosphoramidite with a glycosyl thiol of a protected sialic acid. Compounds 1 and 1S were characterized for their activity in a sialyl transfer assay. The rate of solvolysis in aqueous buffer of analogue 1S was 50-fold slower than that of 1. Analogue 1S was found to be substrate for α-2,3-sialyltransferase. The K_m of 1S was just 3-fold higher than that of 1, while the k_cat of 1S was 2 orders of magnitude lower compared to 1.

Full text of DOI:10.1021/jo000646+

J . Org. Chem. 2000, 65, 6145-6152
6145
Syn th esis a n d Ch a r a cter iza tion of a n An om er ic Su lfu r An a logu e
of CMP -Sia lic Acid
Scott B. Cohen and Randall L. Halcomb*
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
halcomb@colorado.edu
Received April 26, 2000
R-2,3-Sialyltransferase catalyzes the transfer of sialic acid from CMP-sialic acid (1) to a lactose
acceptor. An analogue of 1 was synthesized in which the anomeric oxygen atom was replaced with
a sulfur atom (1S). The key step in the synthesis of 1S was a tetrazole-promoted coupling of a
cytidine-5′-phosphoramidite with a glycosyl thiol of a protected sialic acid. Compounds 1 and 1S
were characterized for their activity in a sialyl transfer assay. The rate of solvolysis in aqueous
buffer of analogue 1S was 50-fold slower than that of 1. Analogue 1S was found to be substrate for
R-2,3-sialyltransferase. The Km of 1S was just 3-fold higher than that of 1, while the kcat of 1S was
2 orders of magnitude lower compared to 1.
Sch em e 1
In tr od u ction
Sialic acid is a nine-carbon monosaccharide found at
the termini of many complex oligosaccharides and gly-
coproteins in mammalian systems. Biological functions
of sialic acid include cell-cell recognition, cellular adhe-
sion, and receptor-ligand interactions.1 Sialyltrans-
ferases catalyze the transfer of sialic acid from CMP-sialic
acid (1) to a lactose acceptor to produce sialylated product
2 (Scheme 1). The reaction proceeds through a nucleo-
philic displacement with inversion of configuration at the
anomeric center, with cytidine monophosphate (CMP) as
the leaving group. Numerous sialyltransferases have
been identified, each displaying unique substrate speci-
ficity.2
Our interest in sialyltransferases lies in their synthetic
utility to sialylate oligosaccharides and glycoconjugates
as an alternative to traditional chemical glycosylation.
Enzymatic sialylation proceeds regio- and stereoselec-
tively in aqueous solution without the need for tedious
protecting group schemes. Chemical glycosylation with
sialic acid is particularly difficult as a result of the
presence of a carboxylate at the anomeric center. Con-
sequently, the anomeric position is sterically hindered,
and the electron-withdrawing nature of the carboxylate
disfavors formation of an oxocarbenium ion at the ano-
meric carbon. Sialyltransferases have already seen some
utility in the synthesis of oligosaccharides.3 Unfortu-
nately, the low catalytic constants (kcat) for these en-
zymes, on the order of 102 min-1, necessitate a time scale
of days for preparative sialylations. A significant improv-
ment in the synthetic utility of sialyltransferases would
be achieved by understanding and potentially accelerat-
ing their reaction. We have therefore undertaken the
synthesis and evaluation of an anomeric sulfur analogue
(1S, Figure 1) of CMP-sialic acid (1) as a potential
substrate for R-2,3-sialyltransferase. Analogue 1S ap-
peared as a promising candidate for an active sialyl-
transferase substrate given that phosphorothioates in
general are superior leaving groups compared to phos-
phates.4 While our primary goal of accelerating the
transferase reaction has not yet been realized, this report
presents a convergent synthesis of this unique sialyl
conjugate and its activity in a sialyltransfer assay.
Resu lts a n d Discu ssion
Syn th esis of Su lfu r An a logu e 1S. A general route
for the synthesis of CMP-sialic acid and CMP-conjugates
containing modified sialic acids has been developed in
our laboratory.5 The synthesis of 1S expands on this
chemistry and demonstrates its application for the
construction of phosphorothioate analogues of 1. The key
step in the synthesis is the coupling of a protected
cytidine-5′-phosphoramidite with a glycosyl thiol of a
(1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Schauer, R.; Kamerling,
J . P. In Glycoproteins II; Montreuil, J ., Vliegenhart, J . F. G., Schachter,
H., Eds.; Elsevier: Amsterdam, 1997; pp 243-402.
(2) Williams, M. A.; Hiroshi, K.; Datta, A. K.; Paulson, J . C.;
J amieson, C. J . Glycoconjugate J . 1995, 12, 755.
(3) (a) Chappell, M. D.; Halcomb, R. L. J . Am. Chem. Soc. 1997,
119, 3393. (b) Halcomb, R. L.; Huang, H.; Wong, C. J . Am. Chem. Soc.
1994, 116, 11315. (c) Wong, C.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Elsevier: New York, 1994; Chapter 5. (d) Liu, K.;
Danishefsky, S. J . J . Am. Chem. Soc. 1993, 115, 4933. (e) Sabesan, S.;
Paulson, J . C. J . Am. Chem. Soc. 1986, 108, 2068.
(4) Frey, P. A.; Sammons, R. D. Science 1985, 228, 541.
(5) Chappell, M. D.; Halcomb, R. L. Tetrahedron 1997, 53, 11109.
10.1021/jo000646+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/22/2000

6146 J . Org. Chem., Vol. 65, No. 19, 2000
Cohen and Halcomb
Sch em e 3
F igu r e 1. Structures of CMP-sialic acid (1) and the anomeric
sulfur analogue (1S) characterized in this work.
Sch em e 2
Tetrazole-promoted activation of phosphoramidite 58
in the presence of glycosyl thiol 8 at -40 °C cleanly
afforded the corresponding thiophosphite product (Scheme
4). In early experiments, this P(III) product was isolated
but was prone to oxidation by ambient molecular oxygen
during workup and purification (ca. 25% of product
oxidized). Therefore, the thiophosphite was not isolated
but rather oxidized in situ at -40 °C with dimethyldiox-
irane (Scheme 4).9 Purification over lipophilic sephadex
(Sephadex LH-20, CH3CN as mobile phase) afforded the
phosphorothioate triester 9 in 80% yield as a 2:1 mixture
of diastereomers. The use of the O-allyl phosphate
protecting group allowed deallylation of 9 under neutral
conditions by palladium(0)-catalyzed allyl ester exchange
with potassium 2-ethylhexanoate (Scheme 4).10 Sialyl
conjugates such as 9 are not compatible with the more
common base-labile phosphate protecting groups such as
â-cyanoethyl because conditions for removing these groups
can lead to elimination across the C2-C3 bond of the
sialic acid. In contrast, anionic phosphate diesters such
as 10 are relatively robust, allowing for smooth deacy-
lation with sodium methoxide and final methyl ester
saponification with sodium hydroxide to afford sulfur
protected sialic acid. The cytidine phosphoramidite was
synthesized in four steps beginning with selective protec-
tion of the 5′-alcohol of cytidine as the TBDPS ether
(Scheme 2). Benzoylation of the 2′- and 3′-alcohols and
the C4-amine was followed by liberation of the 5′-alcohol
with tetrabutylammonium fluoride to provide 4. Phos-
phitylation with O-allyl tetraisopropylphosphordiamidite
afforded the cytidine O-allyl phosphoramidite 5.
Incorporation of the sulfur at the anomeric position of
sialic acid employed Lewis acid catalyzed glycosylation
with a sulfur nucleophile (Scheme 3). Peracetylated sialic
acid methyl ester was treated with anhydrous HF-
pyridine as described by Olah and co-workers6 to afford
the â-fluoride 6. Activation of 6 with boron trifluoride in
the presence of excess thiolacetic acid for 12 h afforded
the thiolacetate 7 with good â-selectivity (â:R ≈ 20:1,
determined by 1H NMR).7 The initial addition of thiolace-
tic acid occurs within minutes to afford a kinetic distri-
bution of anomers (â:R ≈ 1:1); the products equilibrate
under the reaction conditions over several hours to give
the thermodynamically more stable â product, as ex-
pected by the anomeric effect. Selective S-deacylation was
achieved with sodium methoxide at -40 °C to provide 8
(Scheme 3).
1
analogue 1S. The H NMR spectrum of 1S bears marked
similarity to that of 1, including phosphorus coupling to
the axial proton of the C3 methylene. The 31P NMR
spectrum displays a sharp singlet at δ 14.73 ppm (H3PO4
) 0 ppm), consistent with data from bridging phospho-
rothioate diesters reported in RNA systems.11
Sia lyl Tr a n sfer Assa yed by HP LC. Activity assays
for sialyltransferases typically monitor the consumption
of a radiolabeled CMP-sialic acid substrate. Consumption
of 1 does not accurately reflect the formation of sialylated
product, but rather is a composite of four different
reactions: (1) solvolysis of 1 free in solution to produce
sialic acid and CMP, (2) enzyme-catalyzed solvolysis to
produce sialic acid and CMP, (3) enzyme-catalyzed sol-
volysis to produce sialyl phosphate and cytidine, (4) the
(8) Barone, A. D.; Tang, J .; Caruthers, M. H. Nucleic Acids Res.
1984, 12, 4051.
(9) Chappell, M. D.; Halcomb, R. L. Tetrahedron Lett. 1999, 40, 1.
(10) J effrey, P. D.; McCombie, S. W. J . Org. Chem. 1982, 47, 587.
(11) Li, X.; Scott, G. K.; Baxter, A. D.; Taylor, R. J .; Vyle, J . S.;
Cosstick, R. J . Chem. Soc., Perkin Trans. 1 1994, 2123.
(6) Olah, G. A.; Nojima, M.; Kerekes, I. Synthesis 1973, 779.
(7) Preparation of 7 from the R-fluoride has been reported: Kanie,
O.; Nakamura, J .; Kiso, M.; Hasegawa, A. J . Carbohydr. Chem. 1987,
6, 105. A synthesis of the R anomer of 1S has been reported: Smalec,
B.; von Itzstein, M. Carbohydr. Res. 1995, 266, 269.

Anomeric Sulfur Analogue of CMP-Sialic Acid
J . Org. Chem., Vol. 65, No. 19, 2000 6147
Sch em e 4
Sch em e 5
F igu r e 2. A rp-HPLC trace of the reaction of 1 (0.5 mM) and
13 (20 µM) with R-2,3-ST (20 mU/mL) monitored by measuring
absorption at 348 nm. The reaction was performed at 37 °C in
MES buffer (200 mM, pH 6.0) with NaCl (100 mM), disodium-
EDTA (0.5 mM), and Triton X-100 (0.01%). The reaction was
injected directly onto the HPLC after 2 h reaction time to
afford 68% 14.
cytosine chromophore (λmax at 272 nm). The transfer of
sialic acid from 1 to 13 catalyzed by R-2,3-sialyltrans-
ferase (R-2,3-ST) is cleanly resolved with reversed-phase
HPLC (rp-HPLC) down to 20 µM 13 (Figure 2). The
identity of the sialylated product as 14 was confirmed
sialyl transfer reaction of interest. The alternative to
monitoring consumption of 1 is to quantitate the radio-
labeled sialylated product after its isolation by size-
exclusion chromatography.12 For this work, a HPLC
assay was developed to allow rapid quantitation specif-
ically of the transfer of sialic acid without the require-
ment of a radiolabeled substrate. A lactose acceptor
bearing a UV-chromophore was synthesized in two steps
from the lactose trichloroacetimidate 11 (Scheme 5).13
Standard borontrifluoride-catalyzed glycosylation14 of 11
with 4,5-dimethoxy-2-nitrobenzyl alcohol afforded 12
exclusively as the â-anomer. Deacylation of 12 with
sodium methoxide gave the water-soluble lactose acceptor
13, which displayed a λmax at 348 nm (ꢀ348 ) 6000 M-1
cm-1). Such a UV absorption is far removed from the
1
by H NMR and high-resolution mass spectrometry of a
preparative-scale reaction product.
Solvolysis of 1S. The first characterization of 1S with
respect to 1 was the solvolysis in aqueous solution.
Substrate 1 (0.5 mM) was incubated at 37 °C in HEPES
buffer (100 mM, pH 7.5) or MES buffer (100 mM, pH 6.0)
with sodium chloride (100 mM) and disodium-EDTA (0.5
mM), and its transformation to CMP and sialic acid was
monitored by rp-HPLC. The solvolysis of 1 displayed first-
order behavior and increased in rate upon lowering the
pH from 7.5 to 6.0 (Figure 3). The same experiment with
1S conducted in parallel revealed an unexpectedly slow
rate of solvolysis. Substrate 1S displayed almost 2 orders
of magnitude slower solvolysis than 1 (Figure 3). As
observed for 1, solvolysis of 1S was also accelerated by
lowering the pH of the reaction. The stability of 1S was
(12) Weinstein, J .; Souza-e Silva, U.; Paulson, C. J . J . Biol. Chem.
1982, 257, 13845.
(13) Zollo, P.; Sina¨y, P. Carbohydr. Res. 1986, 150, 199.
(14) Schmidt, R. R. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21.

6148 J . Org. Chem., Vol. 65, No. 19, 2000
Cohen and Halcomb
Ta ble 1. Kin etic a n d Th er m od yn a m ic P a r a m eter s for
Tr a n sfer a se Su bstr a tesa
substrate
Km (mM)
kcat (min-1
)
1
1S
0.3 ( 0.1
0.9 ( 0.2
170 ( 20
2.0 ( 0.2
a
Reactions conducted at 37 °C in MES buffer (200 mM, pH 6.0)
with NaCl (100 mM), disodium-EDTA (0.5 mM), Triton X-100
(0.01%), 13 (20 µM), and R-2,3-ST (20 mU/mL for 1, 100 mU/mL
for 1S). The concentration of transferase substrate was varied from
20 to 1000 µM. Each value is the mean ( range of three
independent determinations.
observed for the catalytic constants. Replacement of
oxygen with sulfur greatly decreased the rate of transfer.
Thus, although 1S is a substrate for R-2,3-ST, it appears
to be significantly less reactive than the natural substrate
(1).
F igu r e 3. Reaction profiles for the solvolysis of 1 and 1S in
aqueous solution. Reactions were performed at 37 °C with
either 1 (0.5 mM) or 1S (0.5 mM) in HEPES buffer (100 mM,
pH 7.5) or MES buffer (100 mM, pH 6.0) with NaCl (100 mM)
Su m m a r y
and disodium-EDTA (0.5 mM): (9) 1, pH ) 7.5, k ) 0.032 h-1
;
In summary, this work expands on a general synthetic
route to CMP-sialic acid conjugates5 to prepare an
anomeric sulfur analogue (1S) of CMP-sialic acid (1).
Although 1S is a substrate for R-2,3-ST, it is 2 orders of
magnitude less reactive toward sialyl transfer than 1.
One possible implication from these results is that the
reaction of 1 with R-2,3-ST requires initial protonation
of the anomeric oxygen atom by the enzyme. Sulfur,
carrying less basic lone electron pairs than oxygen, would
be slower to protonate. While this hypothesis is consistent
with the apparent reactivity of 1S, it cannot be concluded
unequivocally from the limited data presented here.
Given the remarkable stability of 1S in conjuction with
its competent binding, it could possibly find use as a
relatively inert substrate analogue in ongoing efforts to
obtain X-ray structures of sialyl transferases.
(b) 1, pH ) 6.0, k ) 0.13 h-1; (0) 1S, pH ) 7.5, k ) 0.0006
h-1; (O) 1S, pH ) 6.0, k ) 0.003 h-1
.
surprising, considering that replacing the anomeric
oxygen atom with sulfur should result in a better leaving
group in the form of a phosphorothioate, with the sulfur
bearing a negative charge.4
Sia lyl Tr a n sfer Activity of 1S. Sialyl transfer assays
were conducted at 37 °C in aqueous MES buffer (200 mM,
pH 6.0) with sodium chloride (100 mM), disodium-EDTA
(0.5 mM, the STs do not require divalent metal cofac-
tors),15 and lactose acceptor 13 (20 µM). With a saturating
excess of 1 (0.5 mM), transformation of 13 to 14, as
monitored by rp-HPLC (Figure 2), displayed first-order
behavior and afforded a final end point of 95% 14. Sulfur
analogue 1S was also observed to be a substrate for R-2,3-
ST, affording the same product 14. This structural
assignment was confirmed by comparison of the product
with the authentic sample of 14 prepared from 1; the two
compounds were found to be identical in terms of HPLC
retention time (co-injection), UV-absorption spectra, and
mass spectra of the product collected directly from the
HPLC [MALDIMS calculated for (M + Na)+ 851; found
851].
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Ma ter ia ls. Authentic CMP-
sialic acid (1, disodium salt) was obtained from CalBiochem
at a purity of 91%, the impurities being CMP (7%) and cytidine
(2%). Aliquots of 1 were prepared in aqueous HEPES buffer
(10 mM, pH 7.5) at a concentration of 5 mM and stored frozen
at -20 °C; aliquots were consumed within 2 weeks. Rat liver
R-2,3-ST was obtained from CalBiochem at a concentration of
1 mU/µL. The enzyme was stored at -20 °C and consumed
within 2 weeks.
Proton NMR (1H NMR) spectra were recorded at 500 MHz.
Chemical shifts are expressed in parts per million (δ) and are
referenced to residual protium in the NMR solvent: CD3S(O)-
CD2H, δ 2.49; CD2HOD, δ 3.31; DOH, δ 4.80; C6D5H, δ 7.16.
Carbon NMR (13C NMR) spectra were recorded at 125 MHz.
Chemical shifts (δ ppm) are referenced to the carbon signal
for the solvent: DMSO-d6, 39.51; C6D6, 128.39; carbon spectra
recorded in D2O are referenced to an external standard of
DMSO-d6. Fluorine NMR (19F NMR) spectra were recorded at
470 MHz and are referenced to an internal standard of C6H5F
(δ 0.00). Phosphorus NMR (31P NMR) spectra were recorded
at 202 MHz and are referenced to an external standard of 1%
aqueous H3PO4 (δ 0.00).
Michaelis-Menton (Km) and catalytic constants (kcat
)
for substrates 1 and 1S were determined using initial
rate velocities as a function of substrate concentration
(Table 1).16 The apparent binding affinity of 1S for R-2,3-
ST was just 3-fold lower than that of 1. This modest
decrease may be attributed to the slightly longer bond
lengths and smaller bond angles of sulfur compared to
oxygen (e.g., for methanol, C-O ) 1.43 Å, C-O-H )
109°; for methanethiol, C-S ) 1.82 Å, C-S-H ) 100°).17
A much larger effect, nearly 2 orders of magnitude, was
2′,3′-O,N4-Tr iben zoyl-5′-O-ter t-bu tyld ip h en ylsilyl Cy-
tid in e (3). Cytidine (6.0 g, 25 mmol, 1.0 equiv) and imidazole
(4.2 g, 63 mmol, 2.5 equiv) were dissolved in dimethylforma-
mide (45 mL). tert-Butyldiphenylsilyl chloride (7.0 mL, 27
mmol, 1.1 equiv) was added dropwise over a period of 10 min.
The reaction was stirred at room temperature for 1 h and then
quenched by the addition of methanol (10 mL). The solvents
were removed by rotary evaporation at 40 °C under reduced
pressure. The resulting syrup was partitioned between water
and dichloromethane. The aqueous layer was further extracted
(15) Brunner, M.; Horenstein, B. A. Biochemistry 1998, 37, 289.
(16) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H.
Freeman and Co.: New York, 1985; Chapter 3.
(17) CRC Handbook of Chemistry and Physics, 66th ed.; Weast, R.
C., Ed; CRC Press: Boca Raton, FL, 1986; p F-169.

Anomeric Sulfur Analogue of CMP-Sialic Acid
J . Org. Chem., Vol. 65, No. 19, 2000 6149
with dichloromethane. The organic extracts were combined,
and upon standing, the silylated product crystallized from
solution within ca. 10 min. The white crystals were filtered
and dried under vacuum. The crystalline product was dissolved
in pyridine (40 mL, 500 mmol, 20 equiv). Benzoic anhydride
(56 g, 250 mmol, 10 equiv) was added, and the reaction was
stirred at room temperature for ca. 2 d until TLC (SiO2, 1/1
hexane/ethyl acetate) indicated a single product (Rf ) 0.30).
The reaction was quenched with water (20 mL), and the
solvents were removed by rotary evaporation at 40 °C under
reduced pressure. The resulting syrup was partitioned between
water and dichloromethane. The aqueous layer was further
extracted with dichloromethane, and the combined organic
extracts were washed with 5% aqueous HCl, saturated aque-
ous sodium bicarbonate, and saturated aqueous sodium chlo-
ride. The organic layer was dried (Na2SO4), filtered, and
concentrated. The product was purified over silica gel (300
mL), eluting with 6/4 hexane/ethyl acetate to 1/1 hexane/ethyl
acetate. The product was obtained as a white foam (15.8 g,
145.59, 135.61, 133.87, 132.83, 129.32, 128.75, 128.45, 123.51,
115.53, 115.24, 96.69, 89.19, 81.70, 74.32, 71.42, 63.91, 42.46,
24.35. 31P NMR (DMSO-d6, H3PO4 reference): δ 148.34, 148.24.
IR (cm-1): 3069, 2968, 1733. HRFABMS: calcd for (M + H)+,
743.2846; found, 743.2869.
Meth yl(5-Acetam ido-4,7,8,9-tetr a-O-acetyl-3,5-dideoxy-2-
flu or o-â-D-glycer o-D-galacto-2-n on u lopyr an osid)on ate (6).
Sialic acid (3.0 g, 9.7 mmol, 1.0 equiv) was dissolved in pyridine
(16 mL, 195 mmol, 20 equiv). Acetic anhydride (14 mL, 145
mmol, 15 equiv) was added, and the reaction was stirred at
room temperature for 2 h. The solvents were removed by rotary
evaporation at 40 °C under reduced pressure. The resulting
syrup was dissolved in methanol (100 mL), and cesium
carbonate (4.1 g, 12.6 mmol, 1.3 equiv) was added. After
dissolution and CO2 evolution had ceased, the solution was
concentrated under vacuum. The syrup was dissolved in
dimethylformamide (15 mL), and iodomethane (12 mL, 195
mmol, 20 equiv) was added. The reaction was stirred at room
temperature for 24 h and then concentrated by rotary evapo-
ration at 40 °C under reduced pressure. The crude mixture
was partitioned between water and dichloromethane. The
aqueous layer was further extracted with dichloromethane,
and the combined organic extracts were dried (Na2SO4),
filtered, and concentrated to a volume of ca. 20 mL. The
solution was transferred to a 50-mL polypropylene centrifuge
tube and concentrated to a minimum volume (ca. 8 mL) under
a stream of nitrogen. The tube was placed in an ice-water
bath, and hydrogen fluoride-pyridine (10 mL, 70% HF) was
added. After stirring for 15 min at 0 °C, the icebath was
removed, and the reaction was stirred at room temperature
for 4 h. The reaction solution was then transferred carefully
with a syringe to a 1-L flask containing cold saturated aqueous
sodium bicarbonate (600 mL) and dichloromethane (200 mL)
with vigorous stirring. After neutralization, the mixture was
transferred to a 1-L separatory funnel, and the aqueous layer
was further extracted with dichloromethane. The combined
organic extracts were dried (Na2SO4), filtered, and concen-
trated. The glycosyl fluoride was purified over silica gel (200
mL), eluting with 6/4 ethyl acetate/hexane to 8/2 ethyl acetate/
hexane. The product was obtained as a clear, colorless oil (3.4
1
80%). H NMR (DMSO-d6): δ 11.38 (s, 1 H), 8.31 (d, J ) 7.4
Hz, 1 H), 8.02 (m, 2 H), 7.87 (m, 3 H), 7.68-7.60 (m, 7 H),
7.54-7.30 (m, 14 H), 6.28 (d, J ) 2.9 Hz, 1 H), 5.91 (m, 2 H),
4.61 (q, J ) 3.9, 5.2 Hz, 1 H), 4.11 (dd, J ) 3.3, 11.5 Hz, 1 H),
3.97 (dd, J ) 4.1, 11.7 Hz, 1 H), 1.01 (s, 9 H). IR (cm-1): 3068,
2918, 1731. HRFABMS: calcd for (M + H)+, 794.2898; found,
794.2930.
2′,3′-O,N4-Tr iben zoyl Cytid in e (4). The protected cytidine
derivative 3 (15.5 g, 19.5 mmol, 1.0 equiv) was dissolved in
tetrahydrofuran (30 mL). Acetic acid (1.7 mL, 29 mmol, 1.5
equiv) was added, followed by tetrabutylammonium fluoride
solution (1.0 M in THF, 59 mL, 59 mmol, 3.0 equiv). The
reaction was stirred at room temperature for ca. 1 h until TLC
(SiO2, 1/1 hexane/ethly acetate) indicated complete conversion
to a polar product (Rf ) 0.10). The solvent was removed by
rotary evaporation, and the resulting syrup was partitioned
between saturated aqueous sodium bicarbonate and dichlo-
romethane. The aqueous layer was further extracted with
dichloromethane, and the combined organic extracts were
washed with saturated aqueous sodium chloride. The organic
layer was dried (Na2SO4), filtered, and concentrated. The 5′-
alcohol was purified over silica gel (300 mL), eluting first with
1/1 hexane/ethyl acetate to remove the tert-butyldiphenylsilyl
fluoride, followed by 100% ethyl acetate to elute the product.
To the product solution was slowly added approximately equal
volume hexane to afford a white crystalline product. The white
crystals were filtered and dried under vacuum (10.5 g, 95%).
Mp ) 180-182 °C. 1H NMR (DMSO-d6): δ 11.31 (s, 1 H), 8.52
(d, J ) 7.0 Hz, 1 H), 8.02 (d, J ) 7.5 Hz, 2 H), 7.93 (d, J ) 7.6
Hz, 2 H), 7.83 (d, J ) 7.4 Hz, 2 H), 7.68-7.58 (m, 3 H), 7.53-
7.39 (m, 7 H), 6.38 (d, J ) 5.2 Hz, 1 H), 5.85 (t, J ) 5.3 Hz, 1
H), 5.79 (t, J ) 5.6 Hz, 1 H), 5.50 (t, J ) 5.1 Hz, 1 H), 4.53 (q,
J ) 3.2, 4.0 Hz, 1 H), 3.88 (m, 1 H), 3.81 (m, 1 H). 13C NMR
(DMSO-d6): δ 167.39, 164.73, 164.49, 163.62, 154.55, 145.73,
133.84, 132.81, 129.29, 128.82, 128.45, 96.87, 88.46, 83.19,
74.43, 71.49, 60.52. IR (cm-1): 3313, 3063, 2930, 1728. HR-
FABMS: calcd for (M + H)+, 556.1720; found, 556.1694.
3-P r op en yl-(2′,3′-O,N4-tr iben zoyl Cytid in e-5′)-yl-N,N′-
d iisop r op ylp h osp h or a m id ite (5). The 5′-alcohol 4 (1.8 g,
3.2 mmol, 1.0 equiv) and tetrazole-diisopropylammonium salt
(0.28 g, 1.6 mmol, 0.5 equiv) were dissolved in dichloromethane
(10 mL). Allyl-N,N,N′,N′-tetraisopropylphosphordiamidite (1.75
mL, 5.4 mmol, 1.7 equiv) was added, and the solution stirred
at room temperature for ca. 4 h until TLC (SiO2, 1/1 hexane/
ethyl acetate) indicated complete conversion of the 5′-alcohol
to a less polar product (Rf ) 0.30). The reaction solution was
concentrated to a volume of ca. 5 mL and then purified directly
over silica gel (250 mL), eluting with 1/1 hexane/ethyl acetate.
The 5′-phosphoramidite was obtained as a clear, colorless oil
1
g, 70%). TLC (SiO2, 100% ethyl acetate): Rf ) 0.35. H NMR
(C6D6): δ 5.64 (d, J ) 10.1 Hz, 1 H), 5.62 (dd, J ) 2.4, 6.3, 1
H), 5.54 (td, J ) 2.6, 6.3 Hz, 1 H), 5.19 (td, J ) 4.5, 11.3, 1 H),
4.75 (dd, J ) 2.7, 12.5 Hz, 1 H), 4.55 (q, J ) 10.5 Hz, 1 H),
4.26 (dd, J ) 2.6, 8.4 Hz, 1 H), 4.24 (dd, J ) 6.3, 12.5 Hz, 1
H), 3.34 (s, 3 H), 2.40 (dt, J ) 4.5, 13.9 Hz, 1 H), 2.05 (dq, J )
13.8, 36.5 Hz, 1 H), 1.99 (s, 3 H), 1.94 (s, 3 H), 1.79 (s, 3 H),
1.74 (s, 3 H), 1.71 (s, 3 H). 13C NMR (C6D6): δ 170.94, 170.74,
170.61, 170.44, 170.04, 165.24 (d, J ) 29.1 Hz), 108.77 (d, J )
228 Hz), 73.97, 71.11, 68.95, 67.92, 63.04, 53.95, 53.24, 48.81,
36.18 (d, J ) 27.4 Hz), 23.35, 21.15, 20.95, 20.79, 20.76. 19F
NMR (C6D6, C6H5F reference): δ 2.62 (dd, J ) 4.6, 36.6 Hz).
IR (cm-1): 2959, 1748. HRFABMS: calcd for (M + H)+,
494.1674; found, 494.1694. Anal. Calcd for C20H28NO12F: C,
48.68; H, 5.72; N, 2.84. Found: C, 48.64; H, 5.73; N, 2.87.
Meth yl (5-Aceta m id o-4,7,8,9-tetr a -O-a cetyl-2-S-a cetyl-
3,5-d id eoxy-2-t h io-â-D-glycer o-D-ga la ct o-2-n on u lop yr a -
n osid )on a te (7). The glycosyl fluoride 6 (970 mg, 2.0 mmol,
1.0 equiv) was dissolved in dichloromethane (28 mL). Thi-
olacetic acid (0.63 mL, 10 mmol, 5.0 equiv, freshly distilled
under reduced pressure at 2 °C) was added, followed by
borontrifluoride-ethyl etherate (1.22 mL, 10 mmol, 5.0 equiv,
freshly distilled), thus affording the following concentrations:
glycosyl fluoride, 70 mM; thiolacetic acid, 350 mM; borontri-
fluoride-etherate, 350 mM. The reaction was stirred at room
temperature for 12 h and then was quenched by the addition
of saturated aqueous sodium bicarbonate (100 mL). The
mixture was transferred to a separatory funnel, and the
aqueous layer was further extracted with dichloromethane.
The combined organic extracts were dried (Na2SO4), filtered,
and concentrated. The product was purified over silica gel (150
mL), eluting with 6/4 ethyl acetate/hexane to 8/2 ethyl acetate/
hexane. The thiolacetate was obtained as a clear, colorless oil
1
(2.2 g, 95%). H NMR (DMSO-d6): δ 11.38 (s, 1 H), 8.37 (d, J
) 7.7 Hz, 1 H), 8.02 (dd, J ) 1.8, 7.4 Hz, 2 H), 7.92 (dd, J )
1.8, 7.6 Hz, 2 H), 7.84 (dt, J ) 1.4, 8.1 Hz, 2 H), 7.68-7.63 (m,
3 H), 7.54-7.40 (m, 7 H), 6.35 (d, J ) 4.4 Hz, 1 H), 5.92 (m, 1
H), 5.82 (m, 2 H), 5.30 (m, 1 H), 5.10 (m, 1 H), 4.68 (m, 1 H),
4.20-3.80 (m, 4 H), 3.58 (m, 2 H), 1.13 (d, J ) 6.8 Hz, 12 H).
13C NMR (DMSO-d6): δ 167.44, 164.61, 164.46, 163.69, 154.39,
1
(700 mg, 65%). TLC (SiO2, 100% ethyl acetate): Rf ) 0.30. H
NMR (C6D6): δ 5.68 (t, J ) 5.5 Hz, 1 H), 5.40 (dt, J ) 2.4, 8.1

6150 J . Org. Chem., Vol. 65, No. 19, 2000
Cohen and Halcomb
Hz, 1 H), 5.21 (dd, J ) 2.2, 12.4 Hz, 1 H), 5.08 (td, J ) 4.5,
11.1 Hz, 1 H), 4.84 (d, J ) 10.3 Hz, 1 H), 4.49 (dd, J ) 7.9,
12.3 Hz, 1 H), 4.45 (q, J ) 10.5 Hz, 1 H), 4.26 (dd, J ) 2.6,
10.5 Hz, 1 H), 3.58 (s, 3 H), 2.50 (dd, J ) 4.8, 13.7 Hz, 1 H),
1.98 (dd, J ) 11.7, 13.7 Hz, 1 H), 1.93 (s, 3 H), 1.86 (s, 3 H),
1.85 (s, 3 H), 1.77 (s, 3 H), 1.65 (s, 3 H), 1.62 (s, 3 H). IR (cm-1):
2956, 1745. HRFABMS: calcd for (M + H)+, 550.1594; found,
550.1594.
-40 °C for 2 h. To oxidize the phosphite, dimethyldioxirane
(100 mM in acetone, 12 mL, 1.2 mmol, 2.5 equiv) was added
at -40 °C, and the reaction was stirred at -40 °C for 1 h.
Excess dimethyldioxirane was quenched at -40 °C by addition
of dimethyl sulfide (0.09 mL, 1.2 mmol, 2.5 equiv). The
suspension was warmed to room temperature and filtered over
Celite. The clear solution was concentrated by rotary evapora-
tion at 30 °C to a volume of ca. 3 mL and immediately purified
over a column of LH-20 sephadex (Sigma) in acetonitrile
(column dimensions, 3 cm × 35 cm; flow rate, 1 drop per 5 s).
The phosphorothioate 9 eluted first. The product, a white oil,
was obtained as a 2:1 mixture of diastereomers (450 mg, 80%).
TLC (SiO2, 8/2 ethyl acetate/hexane): Rf ) 0.10. 1H NMR
(C6D6, major diastereomer): δ 10.10 (br s, 1 H), 8.15-7.95 (m,
7 H), 7.16-6.92 (m, 10 H), 6.30-4.55 (m, 19 H), 3.76 (s, 3 H),
2.72 (br dd, J ) 4.7, 13.5 Hz, 1 H), 2.06 (m, 1 H), 2.03 (s, 6 H),
1.85 (s, 3 H), 1.74 (s, 3 H), 1.61 (s, 3 H). 31P NMR (C6D6, H3-
PO4 reference): δ 21.17 (minor) and 20.79 (major). IR (cm-1):
3069, 1741. EIMS: calcd for (M + H)+, 1165; found, 1165.
Tr ieth yla m m on iu m 2′,3′-O,N4-Tr iben zoyl Cytid in e-5′-
yl (Meth yl (5-acetam ido-4,7,8,9-tetr a-O-acetyl-3,5-dideoxy-
2-t h io-â-D-glyce r o-D-ga la ct o-2-n on u lop yr a n osid -2))-yl
P h osp h or oth ioa te (10). The phosphorothioate triester 9
(325 mg, 0.28 mmol, 1.0 equiv), triphenylphosphine (30 mg,
0.12 mmol, 0.4 equiv), and tetrakis(triphenylphosphine)-
palladium(0) (30 mg, 0.03 mmol, 0.1 equiv) were dissolved in
dichloromethane (10 mL). Deallylation was initiated by addi-
tion of potassium 2-ethylhexanoate (100 mM in ethyl acetate,
3.1 mL, 0.31 mmol, 1.1 equiv). The reaction was stirred for
ca. 30 min until TLC (SiO2, 9/1 dichloromethane/methanol)
indicated complete conversion to a polar product (Rf ) 0.15).
The reaction solution was partitioned between aqueous tri-
ethylammonium bicarbonate (0.1 M) and dichloromethane. The
aqueous layer was further extracted with dichloromethane,
and the combined organic extracts were dried (Na2SO4),
filtered, and concentrated. The product was purified over silica
gel (50 mL), eluting with 96/2/2 dichloromethane/methanol/
triethylamine to afford the triethylammonium salt of 10 as a
white oil (240 mg, 70%). 1H NMR (CD3OD): δ 8.65 (d, J ) 7.5
Hz, 1 H), 8.01 (m, 4 H), 7.88 (dd, J ) 1.2, 8.4 Hz, 2 H), 7.77 (d,
J ) 7.5 Hz, 1 H), 7.63 (m, 2 H), 7.55 (m, 3 H), 7.45 (t, J ) 7.9
Hz, 2 H), 7.37 (t, J ) 7.9 Hz, 2 H), 6.52 (d, J ) 5.4 Hz, 1 H),
5.93 (t, J ) 10.0 Hz, 1 H), 5.87 (t, J ) 11.1 Hz, 1 H), 5.56 (td,
J ) 4.7, 11.1 Hz, 1 H), 5.50 (dd, J ) 2.4, 6.0 Hz, 1 H), 5.29 (td,
J ) 2.5, 6.2 Hz, 1 H), 4.73 (m, 1 H), 4.60 (d, J ) 2.4 Hz, 1 H),
4.58 (t, J ) 2.2 Hz, 1 H), 4.42 (m, 1 H), 4.30 (m, 1 H), 4.22 (dd,
J ) 6.4, 12.3 Hz, 1 H), 3.98 (t, J ) 10.4 Hz, 1 H), 3.81 (s, 3 H),
3.13 (q, J ) 7.4 Hz), 2.96 (dd, J ) 4.7, 13.5 Hz, 1 H), 2.10 (m,
1 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 1.99 (s, 3 H), 1.93 (s, 3 H),
1.83 (s, 3 H), 1.28 (t, J ) 7.4 Hz). 31P NMR (CD3OD, H3PO4
reference): δ 11.30. IR (cm-1): 3420, 2982, 1735. EIMS: calcd
for (M - H)-, 1123; found, 1123.
Disod iu m Cytid in e-5′-yl 5-Aceta m id o-3,5-d id eoxy-2-
th io-â-D-glycer o-D-ga la cto-2-n on u lop yr a n osid -2-yl P h os-
p h or oth ioa te (1S). The protected phosphorothioate diester
10 (triethylammonium salt, 150 mg, 0.12 mmol, 1.0 equiv) was
dissolved in methanol (5 mL). Sodium methoxide (0.5 M in
methanol, 0.70 mL, 0.36 mmol, 3.0 equiv) was added, and the
reaction was stirred at room temperature for 3 h. The reaction
was quenched by addition of ammonium bicarbonate crystals
(47 mg, 0.60 mmol, 5.0 equiv). Upon dissolution of all salts,
the solution was concentrated. The crude product was parti-
tioned between water and ethyl acetate. The aqueous layer
was washed with ethyl acetate and then passed though a
Waters C18 SepPak. The solution was concentrated to a dry
powder by lyophilization, and the resulting methyl ester was
dissolved in aqueous sodium hydroxide solution (0.1 M, 12 mL,
1.2 mmol, 10 equiv). The saponification was allowed to proceed
at room temperature for 30 min and then was quenched by
addition of ammonium bicarbonate crystals (95 mg, 1.2 mmol).
The resulting solution was transferred to a dialysis membrane
(SpectraPor, MWCO ) 100) and dialyzed against aqueous
sodium bicarbonate (10 mM, 4 L) for 48 h. The solution was
concentrated by lyophilization. The product, a white powder,
was obtained as the disodium salt. The yield was determined
Meth yl (5-Acetam ido-4,7,8,9-tetr a-O-acetyl-3,5-dideoxy-
2-th io-â-D-glycer o-D-galacto-2-n on u lopyr an osid)on ate (8).
The thiolacetate 7 (570 mg, 1.04 mmol, 1.0 equiv) was
dissolved in methanol (21 mL), and the solution was cooled to
-40 °C. Sodium methoxide (500 mM in methanol, 2.1 mL, 1.04
mmol, 1.0 equiv, 50 mM final) was added dropwise over a
period of 5 min and the reaction stirred at -40 °C for 30 min.
The reaction was quenched at -40 °C by addition of am-
monium chloride crystals (165 mg, 3.1 mmol, 3.0 equiv) and
the solution was allowed to warm to room temperature. Upon
dissolution of all salts, the solution was concentrated. The
crude product was partitioned between water and dichlo-
romethane. The aqueous layer was further extracted with
dichloromethane, and the combined organic extracts were
dried (Na2SO4), filtered, and concentrated. The product was
purified over silica gel (20 mL), eluting with 8/2 ethyl acetate/
hexane to afford the glycosyl thiol as a clear, colorless oil (420
mg, 80%). TLC (SiO2, 100% ethyl acetate): Rf ) 0.30. 1H NMR
(C6D6): δ 5.67 (dd, J ) 2.1, 4.9 Hz, 1 H), 5.60 (m, 1 H), 5.24
(d, J ) 10.3 Hz, 1 H), 5.18 (td, J ) 4.0, 6.2 Hz, 1 H), 5.05 (dd,
J ) 2.1, 12.3 Hz, 1 H), 4.52 (dd, J ) 2.2, 10.7 Hz, 1 H), 4.43
(q, J ) 10.4 Hz, 1 H), 4.30 (dd, J ) 7.8, 12.5 Hz, 1H), 3.36 (s,
3 H), 2.75 (br s, 1 H), 2.49 (dd, J ) 4.9, 13.9 Hz, 1 H), 2.16
(dd, J ) 11.7, 13.9 Hz, 1 H), 1.97 (s, 3 H), 1.91 (s, 3 H), 1.74 (s,
3 H), 1.72 (s, 3 H), 1.66 (s, 3 H). 13C NMR (C6D6): δ 170.66,
170.65, 170.62, 170.49, 170.10, 84.79, 73.13, 72.58, 69.83,
68.54, 63.50, 53.21, 49.93, 39.05, 23.37, 21.25, 21.05, 20.84.
IR (cm-1): 2961, 1748. HRFABMS: calcd for (M + H)+,
508.1489; found, 508.1493. Anal. Calcd for C20H29NO12S: C,
47.33; H, 5.76; N, 2.76; S, 6.32. Found: C, 47.53; H, 5.78; N,
2.78; S, 6.51.
Dim eth yld ioxir a n e.18 In a 1-L flask equipped with short-
path distilling head and powder addition funnel was vigorously
stirred water (160 mL), acetone (104 mL, spectrophotometric
grade), and sodium bicarbonate (96 g). Oxone (100 g) was
added over a period of 5 min. The system was placed under
vacuum (water aspirator), and the receiving flask (100 mL)
was placed in an acetone/solid CO2 bath. Oxone (100 g) was
added though the addition funnel over a period of 10 min.
Collection continued for another 10 min to afford ca. 30 mL of
a light yellow solution. Calcium sulfate (5 g, finely crushed)
was added as a drying agent. The flask was covered with a
rubber septum and stored at -20 °C vented with a needle to
allow the condensed CO2 to escape. Typically, dimethyldiox-
irane was obtained at a concentration of ca. 100 mM. Titration
was performed on the day of use by measuring the oxidation
of thioanisole by 1H NMR integration of the aromatic reso-
nances.
3-P r open yl (2′,3′-O,N4-Tr iben zoyl Cytidin e-5′)-yl (Meth -
yl (5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-3,5-d id eoxy-2-th io-
â-D-glycer o-D-ga la cto-2-n on u lop yr a n osid -2))-yl P h osp h o-
r oth ioa te (9). The glycosyl thiol 8 (240 mg, 0.48 mmol, 1.0
equiv) and the 5′-phosphoramidite 5 (605 mg, 0.82 mmol, 1.7
equiv) together in
a flask were rendered anhydrous by
concentration from toluene (2 × 5 mL). Activated 3-Å molec-
ular sieves (1 g, finely ground) were added, followed by
acetonitrile (6 mL, distilled first from CaH2, then from P2O5).
The suspension was stirred at room temperature for 1 h and
then cooled to -40 °C. In a second flask were stirred tetrazole
(102 mg, 1.4 mmol, 3 equiv) and activated 3-Å molecular sieves
(0.2 g) in acetonitrile (2 mL) at room temperature for 1 h. The
reaction was initiated at -40 °C by transfer of the tetrazole
solution to the thiol/amidite flask. The reaction was stirred at
(18) Adam, W.; Chan, Y. Y.; Cremer, D.; Gauss, J .; Scheutzow, D.;
Schindler, M. J . Org. Chem. 1987, 52, 2800.

Anomeric Sulfur Analogue of CMP-Sialic Acid
J . Org. Chem., Vol. 65, No. 19, 2000 6151
by UV (ꢀ ) 9200 M-1 cm-1 at 272 nm) (65 mg, 80%). For sialic
acid transfer assays, a solution of 1S in aqueous HEPES buffer
(10 mM, pH 7.5) was prepared at a concentration of 5 mM
and stored until use at -20 °C. 1H NMR (D2O): δ 7.94 (d, J )
7.5 Hz, 1 H), 6.10 (d, J ) 7.5 Hz, 1 H), 5.98 (d, J ) 4.1 Hz, 1
H), 4.32-4.24 (m, 5 H), 4.20-4.10 (m, 2 H), 3.96-3.86 (m, 3
H), 3.61 (dd, J ) 6.9, 11.9 Hz, 1 H), 3.44 (d, J ) 10.0 Hz, 1 H),
2.50 (dd, J ) 4.6, 13.7 Hz, 1 H), 2.04 (s, 3 H), 1.82 (ddd, J )
2.0, 6.4, 13.9 Hz, 1 H). 13C NMR (D2O): δ 176.12, 175.41,
166.86, 161.49, 158.47, 142.41, 97.25, 90.10 (d, J ) 4.4 Hz),
89.63, 83.37 (d, J ) 8.8 Hz), 74.90, 72.69, 70.21, 69.55, 68.21,
65.63 (d, J ) 6.2 Hz), 64.04, 52.76, 43.44 (d, J ) 9.8 Hz), 22.82.
31P NMR (H3PO4 reference): δ 14.73. HRFABMS: calcd for
(M - H2 + Na3)+, 697.0781; found, 697.0810.
(4,5-Dim eth oxy-2-n itr op h en yl)m eth yl O-(2,4,6-Tr i-O-
a ce t yl-â-D-ga la ct op yr a n osyl)-(1f4)-O-2,3,4,6-t e t r a -O-
a cetyl-â-D-glu cop yr a n osid e (12). The lactose trichloroace-
timidate 1113 (270 mg, 0.35 mmol, 1.0 equiv) and 4,5-dimethoxy-
2-nitrobenzyl alcohol (370 mg, 1.8 mmol, 5 equiv) were
dissolved in dichloromethane (20 mL). Borontrifluoride-ethyl
etherate (0.025 mL, 0.18 mmol, 0.5 equiv) was added, and the
reaction was stirred at room temperature for 5 min. The
reaction solution was partitioned between aqueous saturated
sodium bicarbonate and dichloromethane. The organic layer
was dried (Na2SO4), filtered, and concentrated. The product
was purified over silica gel (40 mL), eluting first with 6/4
hexane/ethyl acetate to remove the excess benzyl alcohol
followed by 1/1 hexane/ethyl acetate to elute the product. The
lactose product was obtained as a clear, pale-yellow oil (120
mg, 40%). TLC (SiO2, 1/1 hexane/ethyl acetate): Rf ) 0.10. 1H
NMR (C6D6): δ 7.50 (s, 1 H), 7.17 (s, 1 H), 5.53 (dd, J ) 7.9,
10.6 Hz, 1 H), 5.49 (d, J ) 3 Hz, 1 H), 5.43 (t, J ) 9.3 Hz, 1
H), 5.35 (m, 2 H), 5.13 (dd, J ) 3.4, 10.4 Hz, 1 H), 4.98 (d, J
) 15.0 Hz, 1 H), 4.46 (dd, J ) 1.9, 12.0 Hz, 1 H), 4.38 (d, J )
7.9 Hz, 1 H), 4.34 (d, J ) 7.8 Hz, 1 H), 4.21-4.08 (m, 3 H),
3.71 (t, J ) 9.5 Hz, 1 H), 3.55 (t, J ) 7.0 Hz, 1 H), 3.54 (s, 3
H), 3.29 (m, 1 H), 3.21 (s, 3 H), 1.97 (s, 3 H), 1.96 (s, 3 H), 1.80
(s, 3 H), 1.76 (s, 3 H), 1.75 (s, 3 H), 1.68 (s, 3 H), 1.58 (s, 3 H).
13C NMR (C6D6): δ 170.51, 170.48, 170.29, 170.21, 170.04,
169.65, 169.37, 154.87, 148.82, 139.80, 129.62, 110.37, 108.60,
102.03, 101.01, 77.51, 73.54, 73.46, 72.73, 71.92, 71.28, 70.06,
69.05, 67.40, 62.86, 61.45, 56.27, 55.84, 21.13, 20.87, 20.85,
20.69, 20.59, 20.51. IR (cm-1): 2942, 1752, 1523, 1223. HR-
FABMS: calcd for (M + Na)+, 854.2331; found, 854.2328. Anal.
Calcd. for C35H45NO22: C, 50.54; H, 5.45; N, 1.68. Found: C,
50.60; H, 5.54; N, 1.67.
with a mobile phase of 100% aqueous triethylammonium
bicarbonate buffer (20 mM, pH 8) for 20 min. Sialic acid
transfer assays employed a 50-µL injection loop with a linear
gradient of 80/20 aqueous triethylammonium bicarbonate
buffer (20 mM, pH 8)/methanol at t ) 0 min to 50/50 aqueous
triethylammonium bicarbonate buffer (20 mM, pH 8)/methanol
over a period of 20 min; elution continued for another 10 min
at 50/50 aqueous buffer/methanol. Peaks were detected at 272
nm for solvolysis reactions and 348 nm for transfer assays.
Retention times for reaction components are as follows: CMP,
9 min; 1, 12 min; 14, 20 min; 13, 24 min.
4,5-Dim eth oxy-2-n itr oben zyl O-5-Aceta m id o-3,5-d id e-
oxy-D-glycer o-r-D-ga la cto-2-n on u lop yr a n osylon ic Acid -
(2f3)-O-â-D-Ga la ct op yr a n osyl-(1f4)-â-D-glu cop yr a n o-
sid e (sod iu m sa lt) (14). In a 15-mL polypropylene centrifuge
tube was combined 1 (12 mg), 13 (5 mg), aqueous HEPES
buffer (0.9 mL, 1 M, pH 7.5, sodium salt), aqueous sodium
chloride solution (0.9 mL, 1 M), aqueous disodium-EDTA
solution (0.9 mL, 5 mM, pH 8), aqueous Triton X-100 solution
(0.9 mL, 0.1%), and water (5.0 mL). The reaction was initiated
by addition of R-2,3-ST (0.45 mL, 1 mU/µL), thus affording
the following concentrations of reactants at the onset of the
reaction: 1, 2 mM; 13, 1 mM; HEPES buffer, 100 mM; sodium
chloride, 100 mM; disodium-EDTA, 0.5 mM; Triton X-100,
0.01%; R-2,3-ST, 50 mU/mL. The reaction was incubated at
37 °C for 24 h in the dark. HPLC analysis after 24 h indicated
75% conversion to 14. CMP-sialic acid (6 mg) and R-2,3-ST
(0.45 mL, 1 mU/µL) were added, and the reaction was
incubated for another 24 h in the dark. HPLC analysis after
this time indicated complete conversion of 13 to 14. The
reaction solution was loaded directly onto a column of C18
-
modified reversed-phase silica (Fluka, 400 mesh, column
dimensions 10 mm × 90 mm). Buffers, salts, and cytidine
byproducts were eluted with 30 mL 9/1 aqueous sodium
bicarbonate solution (10 mM)/methanol. Product 14 (yellow)
was then eluted with 7/3 aqueous sodium bicarbonate solution
(10 mM)/methanol. The methanol was removed by rotary
evaporation and the resulting aqueous solution was concen-
trated by lyophilization to afford 14 as a pale-yelleow powder
1
(6 mg, 70%). ꢀ ) 6000 M-1 cm-1 at 348 nm. H NMR (D2O): δ
7.71 (s, 1 H), 7.40 (s, 1 H), 5.20 (d, J ) 15.0 Hz, 1 H), 5.16 (d,
J ) 15.0 Hz, 1 H), 4.57 (d, J ) 8.0 Hz, 1 H), 4.53 (d, J ) 7.9
Hz, 1 H), 4.12 (dd, J ) 3.2, 9.9 Hz, 1 H), 4.00-3.80 (m, 6H),
3.97 (s, 3 H), 3.92 (s, 3 H), 3.78-3.54 (m, 12 H), 3.43 (t, J )
8.7 Hz, 1 H), 2.75 (dd, J ) 4.6, 12.5 Hz, 1 H), 2.03 (s, 3 H),
1.79 (t, J ) 12.2 Hz, 1 H). MALDIMS: calcd for (M + Na)+,
851.2546; found, 851.2543.
(4,5-Dim eth oxy-2-n itr op h en yl)m eth yl O-(â-D-Ga la cto-
p yr a n osyl)-(1f4)-â-D-Glu cop yr a n osid e (13). The lactose
derivative 12 (50 mg, 0.06 mmol, 1.0 equiv) was dissolved in
methanol (5 mL). Sodium methoxide (0.5 M in methanol, 0.24
mL, 0.12 mmol, 2.0 equiv) was added, and the reaction was
stirred at room temperature for 2 h. The reaction was
quenched by addition of ammonium chloride crystals (16 mg,
0.3 mmol, 5 equiv). After dissolution of all salts, the solution
was concentrated. The crude product was dissolved in a
minimum volume of water (ca. 10 mL) and transferred to a
dialysis membrane (SpectraPor, MWCO ) 100). The product
was desalted by dialysis against water (4 L) for 48 h in the
dark. The product was concentrated by lyophilization and
obtained as a pale-yellow powder (29 mg, 90%). For sialic acid
transfer assays, a solution of 13 in water was prepared at a
concentration of 0.2 mM and stored until use at -20 °C (ꢀ )
6000 M-1 cm-1 at 348 nm). 1H NMR (D2O): δ 7.84 (s, 1 H),
7.44 (s, 1 H), 5.24 (d, J ) 15.0 Hz, 1 H), 5.20 (d, J ) 15.0 Hz,
1 H), 4.59 (d, J ) 8.0 Hz, 1 H), 4.46 (d, J ) 7.9 Hz, 1 H), 4.00
(s, 3 H), 3.98 (m, 1H), 3.96 (s, 3 H), 3.93 (d, J ) 3.0 Hz, 1 H),
3.83-3.65 (m, 6 H), 3.60-3.52 (m, 3 H), 3.44 (t, J ) 8.5 Hz, 1
H). MALDIMS: calcd for (M + Na)+, 560.1591; found, 560.1601.
High -P er for m an ce Liqu id Ch om atogr aph y (r p-HP LC).
All solutions in this work were analyzed with a Varian HPLC
solvent delivery system and Varian UV-1 absorbance detector.
All samples were eluted though a Beckman Ultrasphere C18
reversed-phase analytical HPLC column (particle size, 5 µm;
column dimensions, 4.6 mm × 250 mm) at a flow rate of 0.50
mL/min. Solvolysis reactions employed a 10-µL injection loop
Solvolysis of 1 a n d An a logu e 1S. Solvolysis reactions
were performed in a volume of 1.0 mL at 37 °C in 1.5-mL
Eppendorf tubes. CMP-sialic acid (0.10 mL, 5.0 mM) was
added to a solution composed of water (0.60 mL), HEPES
buffer (0.10 mL, 1 M, pH 7.5, sodium salt), aqueous sodium
chloride solution (0.10 mL, 1 M), and aqueous disodium-EDTA
solution (0.10 mL, 5 mM, pH 8), thus affording the following
concentrations of reactants at the onset of the solvolysis
reaction: 1, 0.5 mM; HEPES buffer, 100 mM; sodium chloride,
100 mM; disodium-EDTA, 0.5 mM. The reaction was incubated
at 37 °C. An aliqout of the solvolysis reaction was injected onto
the HPLC at t ) 0. Aliqouts of 50 µL were removed at
appropriate time intervals (see Figure 2), frozen in liquid
nitrogen, and stored frozen until analysis by HPLC. The
percent 1 remaining was calculated relative to t ) 0. The 1
used in this work was obtained at a purity of 91%, containing
CMP (7%) and cytidine (2%). The data were fit to the equation
y ) e-kt, where y is the percent 1 remaining, t is time, and k
is the first-order rate constant. For solvolysis at pH 6.0,
aqueous MES buffer (0.10 mL, 1 M, pH 6.0, sodium salt) was
used in place of HEPES. Solvolysis of the synthetic analogue
1S was performed as described for 1.
Km Deter m in a tion for 1. To each of seven 0.65-mL
Eppendorf tubes was added 200 µL of a solution composed of
water (400 µL), aqueous MES buffer (400 µL, 1 M, pH 6.0,
sodium salt), aqueous sodium chloride solution (200 µL, 1 M),
aqueous disodium-EDTA solution (200 µL, 5 mM, pH 8),
aqueous Triton X-100 solution (200 µL, 0.1%), and an aqueous

6152 J . Org. Chem., Vol. 65, No. 19, 2000
Cohen and Halcomb
solution of 13 (200 µL, 0.2 mM). To each reaction solution was
added a 50-, 25-, 10-, 5-, 3-, 2- or 1-µL aliquot of aqueous 1 (5
mM). Water was then added to afford a final volume of 250
µL for all seven solutions. The reactions were initiated at 37
°C by the addition of R-2,3-ST (5 µL, 1 mU/µL), thus affording
the following concentrations of reactants at the onset of the
reaction: MES buffer, 200 mM; sodium chloride, 100 mM;
disodium-EDTA, 0.5 mM; Triton X-100, 0.01%; 13, 20 µM; 1,
1000, 500, 200, 100, 60, 40, or 20 µM; R-2,3-ST, 20 mU/mL.
Aliquots of 70 µL were removed at 10 and 20 min, frozen in
liquid nitrogen, and stored frozen until analysis by HPLC.
Formation of 14 was less than 15% at 1000 µM 1 and ca. 1%
at 20 µM 1. Initial reaction velocities were obtained by fitting
the data to y ) mx + b, where y is the percent 14, x is time,
and m is the velocity. Velocities were then plotted against [1]
and fitted to the equation y ) m1x/ (m2 + x), where y is the
velocity, x is [1], m1 is [R-2,3-transferase]0 (kcat), and m2 is
Km.
Km Deter m in a tion for Su lfu r An a logu e 1S. To each of
seven 0.65-mL Eppendorf tubes was added 175 µL of a solution
composed of: water (200 µL), aqueous MES buffer (400 µL, 1
M, pH 6.0, sodium salt), aqueous sodium chloride solution (200
µL, 1 M), aqueous disodium-EDTA solution (200 µL, 5 mM,
pH 8), aqueous Triton X-100 solution (200 µL, 0.1%), and an
aqueous solution of 13 (200 µL, 0.2 mM). To each reaction
solution was added a 50-, 25-, 10-, 7.5-, 5-, 3-, or 2-µL aliquot
of aqueous 1S (5 mM). Water was then added to afford a final
volume of 225 µL for all seven solutions. The reactions were
initiated at 37 °C by the addition of R-2,3-ST (25 µL, 1 mU/
µL), thus affording the following concentrations of reactants
at the onset of the reaction: MES buffer, 200 mM; sodium
chloride, 100 mM; disodium-EDTA, 0.5 mM; Triton X-100,
0.01%; 13, 20 µM; 1S, 1000, 500, 200, 150, 100, 60, or 40 µM;
R-2,3-ST, 100 mU/mL. Aliquots of 70 µL were removed at 4
and 8 h, frozen in liquid nitrogen, and stored frozen until
analysis by HPLC. Formation of 14 was less than 15% at 1000
µM 1S and ca. 1% at 40 µM 1S. Initial reaction velocities were
obtained by fitting the data to y ) mx + b, where y is the
percent 14, x is time, and m is the velocity. Velocities were
then plotted against [1S] and fitted to the equation y ) m1x/
(m2 + x), where y is the velocity, x is [1S], m1 is [R-2,3-
transferase]0 (kcat), and m2 is Km.
Ack n ow led gm en t. This research was supported by
the NIH (GM55852). R.L.H. also thanks the National
Science Foundation (Career Award), the Camille and
Henry Dreyfus Foundation (Camille Dreyfus Teacher-
Scholar Award), Pfizer (J unior Faculty Award), and
Novartis (Young Investigator Award) for support. This
work was greatly facilitated by a 500 MHz NMR
spectrometer that was purchased partly with funds from
an NSF Shared Instrumentation Grant (CHE-9523034).
We would like to thank Professor Marvin Caruthers
(University of Colorado, Boulder) for many helpful
discussions regarding phosphoramidite chemistry.
Su p p or tin g In for m a tion Ava ila ble: Copies of represen-
1
tative H and 31P NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O000646+