Substrate specificity and preparative use of recombinant rat ST3Gal III

Article abstract of DOI:10.1081/CAR-120030021

Recombinant rat α(2 → 3)-sialyltransferase (ST3Gal III, EC 2.4.99.6) was expressed from baculovirus infected Sf 9 insect cells in preparative amounts. In order to elucidate substrate tolerances of ST3Gal III, the natural type I and type II substrates 21 a

Full text of DOI:10.1081/CAR-120030021

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Substrate Specificity and Preparative Use of
Recombinant Rat ST3Gal III
Oliver Schwardt a , Gan‐Pan Gao a , Tamara Visekruna a , Said Rabbani a , Ernst Gassmann
b & Dr. Beat Ernst a
a Institute of Molecular Pharmacy, Pharmacenter , University of Basel , Klingelbergstrasse
50, CH‐4056, Basel, Switzerland
b Syngenta Crop Protection AG , Basel, Switzerland
Published online: 18 Aug 2006.
To cite this article: Oliver Schwardt , Gan‐Pan Gao , Tamara Visekruna , Said Rabbani , Ernst Gassmann & Dr. Beat Ernst
(2004) Substrate Specificity and Preparative Use of Recombinant Rat ST3Gal III, Journal of Carbohydrate Chemistry, 23:1,
1-26, DOI: 10.1081/CAR-120030021
To link to this article: http://dx.doi.org/10.1081/CAR-120030021
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 1, pp. 1–26, 2004
Substrate Specificity and Preparative Use of
Recombinant Rat ST3Gal III
Oliver Schwardt,1 Gan-Pan Gao,1 Tamara Visekruna,1 Said Rabbani,1
Ernst Gassmann,2 and Beat Ernst1,
*
1Institute of Molecular Pharmacy, Pharmacenter, University of Basel,
Basel, Switzerland
2Syngenta Crop Protection AG, Basel, Switzerland
ABSTRACT
Recombinant rat a(2!3)-sialyltransferase (ST3Gal III, EC 2.4.99.6) was expressed
from baculovirus infected Sf 9 insect cells in preparative amounts. In order to elucidate
substrate tolerances of ST3Gal III, the natural type I and type II substrates 21 and 22 as
well as several non-natural sugars were investigated. The non-natural Gal-b(1!3)-
Gal(N) (type III) disaccharides 11, 13, and 16 turned out to be surprisingly good
substrates for ST3Gal III. All sialylations were run in preparative scale and kinetic
parameters (Vmax and Km) were determined.
Key Words: ST3Gal III; Gangliosides; CMP-NeuAc; Myelin-associated glycoprotein;
Enzymatic sialylation.
INTRODUCTION
Carbohydrates from glycolipids and glycoproteins with terminal sialic acids are
involved in a broad variety of biological recognition and adhesion phenomena.[1,2]
*
Correspondence: Dr. Beat Ernst, Institute of Molecular Pharmacy, Pharmacenter, University of
Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland; Fax: þ41-61-2671552; E-mail:
beat.ernst@unibas.ch.
1
DOI: 10.1081/CAR-120030021
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Schwardt et al.
Gangliosides, a group of sialylated glycosphingolipids widely present in mammalian
tissues, have been recognized to play an important role in many biological processes
such as cell–cell recognition, cell growth, differentiation, transformation, and neural
functions.[3–6] Thus, the gangliosides GM1b, GD1a, and GT1b (Fig. 1) have been
shown to bind with high affinity to the myelin-associated glycoprotein (MAG), a
member of the siglec family, which is a potent inhibitor of neurite outgrowth of most
neurons.[7–9]
The common element of gangliosides binding to MAG is the terminal NeuAc-
a(2!3)-Gal-b(1!3)-GalNAc partial structure, suggesting a special importance of this
trisaccharide subunit.
In order to investigate their binding affinity to MAG and other siglecs, large
numbers of natural and modified oligosaccharides have to be made available for
extensive biological screening. Although numerous glycosylation methods are known,
the chemical synthesis of complex oligosaccharides is still not a routine procedure.[10]
Figure 1. The gangliosides GM1b, GD1a, and GT1b.

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Recombinant Rat ST3Gal III
3
Difficulties often occur from time-consuming protecting group manipulations.
Cumbersome separation of the desired product from the anomeric mixtures is often
necessary. Finally, chemical sialylations suffer from poor stereoselectivity due to the
lack of neighboring group assistance of the sialic acid.[10] A convenient alternative is
the application of sialyltransferases (STs).[11,12] Depending on their specificity, these
enzymes catalyze the transfer of a sialic acid moiety from cytidine-50-monophospho-N-
acetylneuraminic acid (CMP-NeuAc) a-selectively to the 3-OH or 6-OH of a terminal
galactose residue, or to the 8-OH or 9-OH of a sialic acid unit, depending on the
specificity of the particular enzyme.
The mammalian ST family consists of more than 20 STs, which can be divided into
three sub-families. The members of each sub-family differ in their substrate specificity.
ST8Sia I-V and ST9Sia mediate the transfer of a sialic acid moiety to the 8-OH and 9-OH,
respectively. ST6Gal I and ST6GalNAc I–VI catalyze the a-sialylation of the 6-OH of a
terminal galactose (Gal) residue or a terminal or internal N-acetylgalactosamine group,
respectively.[13,14]
To date, six different a(2!3)-STs have been characterized. ST3Gal I[15] and ST3Gal
II[16] mediate the transfer of sialic acid residues to the galactose unit of terminal Gal-
b(1!3)-GalNAc oligosaccharides found on glycolipids or glycoproteins. Several groups
have reported the successful syntheses of a(2!3)-sialylated O-linked oligosaccharides
employing ST3Gal I.[17] ST3Gal III[18] acts on type I and type II chains [Gal-b(1!3/4)-
GlcNAc] and was, therefore, used for the synthesis of sialyl Lewisa and sialyl Lewisx
epitopes[12,19,20] as well as for the preparation of ganglioside GM3.[21] Recombinant soluble
ST3Gal IV[22] accepts as substrates glycolipids or glycoproteins containing either terminal
Gal-b(1!4)-GlcNAc or Gal-b(1!3)-GalNAc motifs. Recombinant human ST3Gal V was
shown to sialidate only lactosylceramide [Gal-b(1!4)-Glc-b-Cer], whereas the purified rat
liver enzyme exhibited a broader substrate tolerance utilizing also galactosylceramide (Gal-
b-Cer) and asialoganglioside GA2 [GalNAc-b(1!4)-Gal-b(1!4)-Glc-b-Cer] as sub-
strates.[23,24] Finally, human ST3Gal VI[25] accepts almost exclusively the Gal-b(1!4)-
GlcNAc motif on glycoproteins and glycolipids.
Our investigations are aiming at the substrate specificity of STs in general and their
use in preparative scale. In previous studies on the substrate specificity of recombinant
rat ST3Gal III (EC 2.4.99.6), it was found that all of the hydroxyl groups of the
galactose unit are essential for substrate recognition by the enzyme.[26] The enzyme
does, however, accept a wide variety of acyl groups on the glucosamine nitrogen of
type I and type II acceptors.[12,27–29] The replacement of the N-acetyl group by azide,
aromatic or heteroaromatic moieties, charged residues, bulky polar uronic acids or
sulfonamides is well tolerated. Thus, libraries of sialylated type I and type II sugars, the
immediate precursors of the sialyl Lewisa and sialyl Lewisx tetrasaccharides are
produced rapidly with good overall yields. In order to further elucidate the substrate
specificity of recombinant rat ST3Gal III, we investigated in this study its tolerance
towards several type III [Gal-b(1!3)-Gal(N)] disaccharides. The physiological
enzymes for the a(2!3)-sialylation of these sugars are ST3Gal I, II, and IV (EC
2.4.99.4).
Recombinant rat ST3Gal III[30] (The ST3Gal III construct was obtained from
M. Streiff, Novartis Pharma Inc., Basel) was expressed in preparative amounts (9 U/L)
from baculovirus infected Sf 9 insect cells[31] and applied to enzymatic sialylation for
natural and non-natural disaccharides.

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Schwardt et al.
RESULTS AND DISCUSSION
The syntheses of the Gal-GalN and Gal-Gal substrates (type III) are shown in Schs. 1
and 2. Gal-b(1!3)-GalNTCA-b-OSE 11 and Gal-b(1!3)-GalNAc-b-OSE 13 were
easily available from the galactosamide derived building block 8 (Sch. 1).
Compound 8 was synthesized in seven steps from per-O-acetylated N-trichloroacetyl
glucosamide 1[32a] following a procedure by Jacquinet et al.[32b] After the quantitative
transformation of 1 into the corresponding bromide, glycosylation with 2-(trimethylsilyl)-
ethanol (HOSE) was reached in the presence of Hg(CN)2 as promoter to give b-glucoside 2
in 83% yield. Subsequent O-deacetylation afforded 3. Treatment with pivaloyl chloride
(2.5eq. 2308C, pyridine) gave the 3,6-bispivaloate 4 in 89% yield. The 4-hydroxy group
was then transformed into the corresponding triflate, which in the presence of water at 808C
furnished galactosamines 5 and 6 in 83% overall yield.[32b] Transesterification with sodium
methoxide gave 7 (96%), which was transformed into the corresponding benzylidene
derivative 8 with 2,2-dimethoxytoluene in the presence of cat. p-TsOH.
˚
Scheme 1. (a) i. HBr/AcOH, rt, 17hr, ii. HOSE, Hg(CN)2, MS 3 A, toluene, rt, 3 d (89%); (b) cat.
NaOMe/MeOH, rt, 16hr (quant); (c) 2.5eq PivCl, cat. DMAP, pyridine, 2308C, 20 hr (89%); (d) Tf2O,
DCE/pyridine 2 : 1, 08C, 4 hr, then H2O, 808C, 2 hr (83%); (e) 0.1M NaOMe/MeOH, rt, 7 hr, (96%); (f)
PhCH(OMe)2, cat. p-TsOH, MeCN, rt, 4 hr (86%); (g) NIS, TfOH, CH2Cl2, 2258C, 1.5d (64% b); (h) i.
cat. NaOMe/MeOH, rt, 18 hr, ii. 80% aq. AcOH, 808C, 2 hr (46%); (i) Bu3SnH, AIBN, benzene, 808C,
3 hr (93%); (j) i. 80% aq. AcOH, 508C, 4.5hr, ii. cat. NaOMe/MeOH, rt, 21 hr (64%).

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Recombinant Rat ST3Gal III
5
Scheme 2. (a) DMTST, CH2Cl2, 78C, 16 hr (87%); (b) i. NaOMe, MeOH, rt, 2 hr, ii. 10% Pd/C,
H2, MeOH, rt, 3 hr (75%); (c) 10% Pd/C, H2 (4 bar), MeOH/dioxane, rt, 9 d (67%); (d) 18, NIS,
TfOH, CH2Cl2, 2308C, 16 hr (45% a); (e) NaOMe, MeOH, rt, 7 hr, then aq. NaOH, rt, 16 hr (90%).
Galactosylation of 8 using the donor 9[33] promoted by N-iodosuccinimide (NIS)/
TfOH at 2258C gave predominantly disaccharide 10 as 4 : 1-mixture. The separation of
the anomers was achieved by crystallization from i-Pr2O yielding pure b-10 in 64%.
Removal of the benzoyl groups using NaOMe and subsequent cleavage of the benzylidene
group with 80% aqueous acetic acid gave Gal-b(1!3)-GalNTCA-b-OSE (11) in 46%
yield.
For the synthesis of Gal-b(1!3)-GalNAc-OSE (13), the N-trichloroacetyl group in
10 was reduced to the N-acetyl group with Bu3SnH/AIBN in refluxing benzene[32] to give
12 (93%), followed by O-debenzoylation yielding 13 (64%).
Gal-b(1!3)-Gal-b-OSE (16) and Gal-b(1!3)-[NeuAc-a(2!6)]-Gal-b-OSE (20)
were synthesized starting from the known galactose derivative 14[34,35] (Sch. 2).
The coupling of 14[34,35] with donor 9 was promoted by dimethyl(methylthio)
sulfonium triflate (DMTST),[36,37] giving the b-galactoside 15 in excellent yield.
After removal of the benzoate and benzyl protection by transesterification
and hydrogenation, respectively, Gal-b(1!3)-Gal-b-OSE (16) was isolated in 75% yield.
Catalytic hydrogenation of compound 15 turned out to be slow and incomplete,
presumably because of steric hindrance by the benzoyl groups. Therefore, disaccharide 17

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Schwardt et al.
was obtained only in 67%. Sialylation of 17 with the sialic acid donor 18[37] in the
presence of NIS/TfOH afforded the corresponding a-glycoside 19 in 45% yield.
Subsequent saponification of 19 using first sodium methoxide and then sodium hydroxide
gave trisaccharide 20 in 90%.
Following standard sialylation protocols,[12,29,38,39] the saccharides 11, 13, 16, and 20,
and, as control experiments, the natural substrates 21[40] and 22[41] were then incubated in
preparative scale with CMP-NeuAc and recombinant rat ST3Gal III (EC 2.4.99.6, 9 U/L)
(Sch. 3). This enzyme transferred a sialic acid moiety from CMP-NeuAc regio- and
a-stereoselectively onto the 3-OH group of a terminal galactose.
In order to determine the optimal reaction conditions, the activity of the ST3Gal III
over time at 378C was investigated (Fig. 2). Aliquots of the enzyme in sodium cacodylate
buffer were incubated at 378C for 0, 6, 9, 12, 24, 60, and 84 hr, then acceptor 22 and donor
CMP-NeuAc were added and the activity of the ST was determined. As shown in Fig. 2,
the activity of the enzyme remained practically unchanged for the initial 9 hr. Incubation
for 12 and 24 hr, however, led to a significant decrease of activity. It is noteworthy, that
even after 84 hr of incubation at 378C about 20% of the original enzyme activity was still
preserved.
According to these results, the conditions for the reactions in preparative scale were
optimized for the type III disaccharide 13, and were then applied for all the other substrates
(Sch. 3): Substrates and CMP-NeuAc were incubated with ST3Gal III for 3–5 days at
378C in a mixture of 50 mM sodium cacodylate buffer (pH 6.5), 60 mM MnCl2-solution
and water containing BSA and CIAP[21] (EC 3.1.3.1). After 17–24 hr of incubation, an
additional aliquot of transferase was added (except for the natural substrates 21 and 22).
Further addition of ST3Gal III and CMP-NeuAc did not increase the yield of products. The
reactions were monitored by TLC (silica gel, dichloromethane (DCM)/MeOH/H2O
10 : 4 : 0.8). Isolated yields, recovered starting material, and kinetic data (Vmax and Km) are
summarized in Table 1.
As shown before,[29] the natural substrates 21 (type II disaccharide) and 22 (type I)
were converted quantitatively into the corresponding trisaccharides 23 and 24 (entries 1
and 2 in Table 1). Although the physiological enzymes for the sialylation of terminal
Gal-b(1!3)-GalNAc subunits are ST3Gal I, II, and IV (EC 2.4.99.4), compounds 11,
13, and 16 also were sialylated by ST3Gal III giving the trisaccharides 25–27 in
Scheme 3. Enzymatic sialidation using ST3Gal III and CMP-NeuAc.

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Recombinant Rat ST3Gal III
7
Figure 2. Activity over time of ST3 Gal III in the sialidation of Gal-b(1!3)-GlcNAc-b-OLem 22
at 378C.
acceptable yields. In all cases, the unreacted starting materials could be recovered
almost completely. The kinetic data for the sialylation reactions indicate that the
activity of ST3Gal III towards the non-natural substrates 11, 13, and 16 is reduced only
about 10-fold compared to its natural substrates 21 and 22. As expected, the transfer
efficiency Vmax/Km is much lower for the unnatural acceptors as for the natural
substrates 21 and 22. This explains the incomplete, but still preparatively useful,
conversion of the type III disaccharides. 6-O-Sialylated compound 20, however, was
not tolerated as substrate by ST3Gal III, presumably due to the bulky substituent in the
6-position of galactose. Sialylation of a derivative of trisaccharide 20 was recently
reported using the physiological enzyme ST3Gal I.[17e]
Comparative NMR investigations (COSY, HSQC, and TOCSY) of the starting sugars
and the isolated products confirm the attachment of a sialic acid unit. Additional signals in
the 13C NMR spectra at approximately 100 and 40 ppm are characteristic for the C-2 and
C-3 atoms of an a-linked sialic acid.[20a,29,42] Simultaneous down-field shifts (ꢀ4 ppm) of
the galactose C-3 atom in the 13C NMR spectra and about 0.6 ppm of the galactose H-3 in
1
the H NMR spectra confirm the introduction of sialic acid onto the 3-OH group of the
galactose moiety.[29]
In conclusion, our investigations show, in addition to previous reports,[12,29] an
unexpected high substrate tolerance of recombinant ST3Gal III. The enzyme accepts the
replacement of the natural GlcNAc moiety by Gal, GalNAc and GalNTCA. Thus, the
preparative applicability of the transferase has been substantially extended.

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Schwardt et al.

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Recombinant Rat ST3Gal III
9

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Schwardt et al.
EXPERIMENTAL
General Methods
NMR spectra were recorded on a Bruker Avance DMX-500 (500 MHz)
spectrometer. Assignment of 1H and 13C NMR spectra was achieved using 2D
methods (COSY, HSQC, TOCSY). Chemical shifts are expressed in ppm using
residual CHCl3, CHD2OD, and HDO as references. Optical rotations were measured
using a Perkin–Elmer Polarimeter 241. Low resolution LC/MS analyses were carried
out using a Waters Alliance 2690 LC system equipped with a photodiode array
detector and a Micromass Quattro II mass spectrometer. The spectra were recorded in
positive EI mode. The LC method consisted of a separation column (YMC ODS AQ
12.5 cm length, 2 mm i.d.) held at 408C. The mobile phase was MeCN/H2O with the
addition of 0.05% trifluoroacetic acid using a flow rate of 0.5 mL/min. The linear
gradient ran from 0% to 100% MeCN in 15 min followed by 5 min at 100% MeCN
before returning to initial conditions. The LC/HRMS analysis were carried out using
an Agilent 1100 LC equipped with a photodiode array detector and a Micromass
QTOF I equipped with a 4 GHz digital-time converter. All spectra were recorded in
positive EI mode. The LC method consisted of a separation column (YMC ODS AQ
12.5 cm length, 2 mm i.d.) held at rt. The mobile phase was MeCN/H2O with the
addition of 0.5% formic acid using a flow rate of 0.2 mL/min. The linear gradient ran
from 5% to 95% MeCN in 10 min followed by 8 min at 95% MeCN before returning
to initial conditions. Reactions were monitored by TLC using glass plates coated with
silica gel 60 F254 (Merck, CH-8953 Dietikon, Switzerland) and visualized by using
UV light and/or by charring with a molybdate solution (a 0.02 M solution of
ammonium cerium sulfate dihydrate and ammonium molybdate tetrahydrate in
aqueous 10% H2SO4). Column chromatography was performed on silica gel (Uetikon,
40–60 mesh). Methanol (MeOH) was dried by refluxing with sodium methoxide and
distilled immediately before use. Pyridine was freshly distilled under argon over
KOH. Dichloromethane, dichloroethane (DCE), acetonitrile (MeCN), toluene, and
benzene were dried by filtration over Al2O3 (Fluka, CH-9471 Buchs, Switzerland,
˚
type 5016 A basic). Molecular sieves (3 A) were activated in vacuo at 5008C for 2 hr
immediately before use.
Expression of Sialyltransferase ST3Gal III (EC 2.4.99.6)[30,31]
ST3Gal III (EC 2.4.99.6) was expressed using a recombinant baculovirus expression
system. A DNA fragment coding for the chimeric protein containing the untranslated
g-interferon secreting sequence and the sequence for the catalytic domain was cloned into
the commercially available vector pVL1392, a baculovirus expression system transfer
vector (PharMingen). After transfection of Sf-9 cells in monolayer culture with the
recombinant virus (MOI of 20 pfu/cell) soluble a(2!3)-STs were produced.
Complete medium was removed from confluent cells, and fresh medium containing 20
plaque forming units per cell was added. After 1 hr incubation at rt, inoculum was
removed, fresh medium was added, and the culture was returned to the incubator (278C).
Supernatant was collected 72 hr postinfection and spun and filtrated through a membrane

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Recombinant Rat ST3Gal III
11
(0.2 mm). The enzyme activity was measured by the ST activity assay using CMP-[14C]-
NeuAc.
Sialyltransferase Enzyme Assay
The assay mixture (20 mL) containing the acceptor substrate (free disaccharide), ST3Gal
III (0.015mU), 0.1 mM CMP-NeuAc (2nmol) and 60,000cpm of CMP-[14C]-NeuAc in
100 mM sodium cacodylate buffer at pH 6.0 with 0.3% Triton X-100 and 10mM MgCl2 was
incubated at 378C for 1 hr. The reaction mixture was diluted with water to 5 mL and applied
to SepPac (Waters, CH-5102 Rupperswil, Switzerland) C18-cartridges. The column was
washed twice with water (5 mL) and eluted with methanol (6 mL). Incorporation of [14C]-
NeuAc was determined by liquid scintillation counting of the eluate. One unit of activity
throughout this report refers to the amount of enzyme catalyzing the transfer of 1 mmol
NeuAc to the acceptor per min using the standard incubation conditions (378C and pH 6.0).
Determination of the Relative Enzyme Activity at 378C
Aliquots (20 mL) of ST3Gal III (0.015 mU) in 100 mM sodium cacodylate
buffer at pH 6.0 with 0.3% Triton X-100 and 10 mM MgCl2 were incubated at 378C for
0 (control experiment), 6, 9, 12, 24, 60, and 84 hr, respectively. Then the acceptor substrate
Gal-b-(1!3)-GlcNAc-b-OLem (22, 200 mM), CMP-NeuAc (0.1 mmol) and 60,000 cpm
of CMP-[14C]-NeuAc were added and the enzyme activity was determined as described
above. The enzyme activity for each sample was calculated as a percentage relative to the
control experiment (t ¼ 0 hr).
Determination of Kinetic Parameters
Enzyme kinetics of ST3Gal III were determined in eluates as described above.
Relative rates for each substrate were calculated as a percentage of the incorporation of
NeuAc into the D-Gal-containing disaccharide. Under the above conditions, the formation
of products was shown to be linear in time. For the Km value determination 8–9 different
acceptor concentrations were used. Kinetic constants were obtained from double-
reciprocal plots by linear regression analysis of Lineweaver–Burk plots.
2-(Trimethylsilyl)ethyl 3,4,6-tri-O-Acetyl-2-deoxy-2-trichloroacetamido-
b-D-glucopyranoside (2)
To an ice-cooled solution of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trichloroacetamido-D-
glucopyranose 1[32] (2.32 g, 4.71 mmol) in DCM (30 mL) was added dropwise HBr in
acetic acid (4.1 M, 10 mL). After stirring for 17 hr at rt, the mixture was carefully added to
DCM (100 mL) and saturated aqueous KHCO3 (200 mL) under ice-cooling and stirring,
followed by extraction with DCM (3 ꢀ 40 mL). The combined organic layers were dried
over Na2SO4, filtered, and concentrated under reduced pressure. To the residue (2.39 g)

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Schwardt et al.
and 2-(trimethylsilyl)ethanol (1.32 mL, 9.27 mmol) dissolved in toluene, molecular sieves
˚
(3 A, 4 g) and mercuric cyanide (2.34 g, 9.27 mmol) were added. The mixture was stirred at
rt for 3 d under light exclusion. The suspension was filtered through a pad of Celite, which
was washed with toluene (3 ꢀ 20 mL). The combined filtrates were evaporated to dryness
yielding a residue, which was purified by silica gel chromatography (petroleum ether/
EtOAc 2 : 1) to afford 2 (2.28 g, 89%) as a colorless foam.
[a]D 210.5 (c ¼ 0.45, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.00 (s, 9H, SiMe3),
0.93 (m, 2H, H-20), 2.03, 2.04, 2.10 (3s, 9H, 3CH3), 3.58 (m, 1H, H-10a), 3.74 (ddd,
J ¼ 2.5, 4.8, 9.9 Hz, 1H, H-5), 3.90 (dt, J ¼ 8.5, 10.7 Hz, 1H, H-2), 3.97 (m, 1H, H-10b),
4.16 (dd, J ¼ 2.5, 12.3 Hz, 1H, H-6a), 4.29 (dd, J ¼ 4.8, 12.3 Hz, 1H, H-6b), 4.72
(d, J ¼ 8.3 Hz, 1H, H-1), 5.11 (dd, J ¼ 9.3, 9.9 Hz, 1H, H-4), 5.38 (dd, J ¼ 9.3, 10.7 Hz,
1H, H-3), 6.81 (d, J ¼ 8.7 Hz, 1H, NH); 13C NMR (125 MHz, CDCl3): d 21.5 (SiMe3),
18.0 (C-20), 20.6, 20.6, 20.7 (3CH3), 56.2 (C-2), 62.1 (C-6), 67.8 (C-10), 68.5 (C-4), 71.5
(C-3), 71.9 (C-5), 92.3 (CCl3), 99.8 (C-1), 161.8, 169.3, 170.7, 170.8 (4CO); HRMS
(FAB) Calcd for C19H30Cl3NO9Si [M 2 NH4]þ: 567.1099; Found: 567.1087.
2-(Trimethylsilyl)ethyl 2-Deoxy-2-trichloroacetamido-b-D-
glucopyranoside (3)
A solution of 2 (2.27 g, 4.12 mmol) in methanol (15 mL) was treated with 1 M
methanolic NaOMe (1 mL) at rt for 16 hr. The reaction mixture was neutralized with
Dowex 50X8 (Hþ) ion-exchange resin and filtered through a pad of Celite. The Celite was
washed with methanol (3 ꢀ 5 mL), and the combined filtrates were evaporated to dryness.
The crude residue (1.79 g, quant) was used without further purification.
1H NMR (500 MHz, CD3OD): d 0.00 (s, 9H, SiMe3), 0.93 (m, 2H, H-20), 3.27–3.34
(m, 2H, H-4, H-5), 3.58 (ddd, J ¼ 6.1, 9.5, 10.7 Hz, 1H, H-10a), 3.61 (dd, J ¼ 10.4,
10.5 Hz, 1H, H-3), 3.63 (dd, J ¼ 8.0, 10.4 Hz, 1H, H-2), 3.69 (dd, J ¼ 5.5, 12.0 Hz, 1H,
H-6a), 3.89 (dd, J ¼ 2.0, 12.0 Hz, 1H, H-6b), 3.99 (ddd, J ¼ 5.9, 9.6, 10.7 Hz, 1H, H-10b),
4.57 (d, J ¼ 8.0 Hz, 1H, H-1); 13C NMR (125 MHz, CD3OD): d 21.5 (SiMe3), 19.2 (C-20),
59.3 (C-2), 62.8 (C-6), 68.3 (C-10), 72.4 (C-4), 75.3 (C-3), 78.0 (C-5), 101.7 (C-1), 164.4
(CO); HRMS (FAB) Calcd for C13H24Cl3NO6Si [M 2 NH4]þ: 441.0782; Found:
441.0783.
2-(Trimethylsilyl)ethyl 2-Deoxy-3,6-di-O-pivaloyl-2-trichloroacetamido-
b-D-glucopyranoside (4)
To a solution of 3 (2.89 g, 6.80 mmol) and DMAP (20 mg) in pyridine (30 mL) was
added dropwise pivaloyl chloride (2.08 mL, 17.0 mmol) at 2308C. After stirring for 20 hr
at 2308C, the reaction was quenched with methanol (2 mL). After removal of the solvents
under reduced pressure, the residue was redissolved in DCM (100 mL) and the solution
was washed with 5% aqueous HCl (20 mL) and saturated aqueous KHCO3 (20 mL). The
organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was
purified by column chromatography on silica gel (petroleum ether/EtOAc 2 : 1) to give 4
(3.58 g, 89%) as a colorless foam.

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Recombinant Rat ST3Gal III
13
1
[a]D232.6 (c ¼ 0.50, CHCl3); H NMR (500 MHz, CDCl3): d 0.00 (s, 9H, SiMe3),
0.94 (m, 2H, H-20), 1.21, 1.24 (2s, 18H, 2CMe3), 3.25 (d, J ¼ 5.8 Hz, 1H, OH), 3.50–3.60
(m, 2H, H-4, H-10a), 3.61 (ddd, J ¼ 2.5, 5.7, 9.8 Hz, 1H, H-5), 3.90 (ddd, J ¼ 8.3, 9.0,
10.7 Hz, 1H, H-2), 3.94 (m, 1H, H-10b), 4.37 (dd, J ¼ 5.7, 12.1 Hz, 1H, H-6a), 4.44 (dd,
J ¼ 2.4, 12.1 Hz, 1H, H-6b), 4.65 (d, J ¼ 8.3 Hz, 1H, H-1), 5.41 (dd, J ¼ 9.0, 10.7 Hz, 1H,
H-3), 7.05 (d, J ¼ 9.0 Hz, 1H, NH); 13C NMR (125 MHz, CDCl3): d 21.5 (SiMe3), 18.1
(C-20), 27.0, 27.1 (2CMe3), 38.9, 39.0 (2CMe3), 56.0 (C-2), 63.3 (C-6), 67.5 (C-10), 69.9
(C-4), 74.0, 74.1 (C-3, C-5), 92.5 (CCl3), 100.0 (C-1), 161.9, 179.1, 179.9 (3CO); HRMS
(FAB) Calcd for C23H40Cl3NO8Si [M 2 NH4]þ: 609.1933; Found: 609.1909.
2-(Trimethylsilyl)ethyl 2-Deoxy-4,6-di-O-pivaloyl-2-trichloroacetamido-
b-D-galactopyranoside (5) and 2-(Trimethylsilyl)ethyl 2-Deoxy-3,4-di-O-
pivaloyl-2-trichloroacetamido-b-D-galactopyranoside (6)
Trifluoromethane sulfonic anhydride (1.48 mL, 8.99 mmol) was added at 08C to a
solution of 4 (3.55 g, 5.99 mmol) and DMAP (20 mg) in pyridine (7 mL) and DCE
(25 mL). After stirring for 4 hr at this temperature, water (3 mL) was added and the mixture
was stirred for additional 2 hr at 808C. After cooling to rt, DCM (25 mL) was added,
followed by washing with 5% aqueous HCl (20 mL) and saturated aqueous KHCO3. The
organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (petroleum ether/EtOAc,
gradient 3 : 1 to 1 : 1) to give 4,6-dipivaloate 5 (2.52 g, 71%) and 3,4-dipivaloate 6
(422 mg, 12%) as colorless foams.
5: [a]D 213.8 (c ¼ 0.94, CHCl3); 1H NMR (500MHz, CDCl3): d 0.00 (s, 9H, SiMe3),
0.96 (m, 2H, H-20), 1.19, 1.26 (2s, 18H, 2CMe3), 2.78 (d, J ¼ 4.3 Hz, 1H, OH), 3.55 (ddd,
J ¼ 6.4, 8.3, 10.7Hz, 1H, H-2), 3.57 (m, 1H, H-10a), 3.94 (m, 1H, H-5), 3.97 (m, 1H, H-10b),
4.11 (dd, J ¼ 6.3, 11.2Hz, 1H, H-6a), 4.18 (dd, J ¼ 7.3, 11.2 Hz, 1H, H-6b), 4.40 (ddd,
J ¼ 3.7, 4.2, 10.7Hz, 1H, H-3), 4.82 (d, J ¼ 8.3 Hz, 1H, H-1), 5.31 (dd, J ¼ 1.2, 3.7 Hz, 1H,
H-4), 6.91 (d, J ¼ 6.3 Hz, 1H, NH); 13C NMR (125MHz, CDCl3): d 21.5 (SiMe3), 18.2 (C-
20), 27.1, 27.2 (2CMe3), 38.7, 39.3 (2CMe3), 57.2 (C-2), 61.7 (C-6), 67.7 (C-10), 68.8 (C-3),
69.2 (C-4), 71.2 (C-5), 92.3 (CCl3), 98.9 (C-1), 162.7, 177.9, 178.5 (3CO); HRMS (FAB)
Calcd for C23H40Cl3NO8Si [M 2 NH4]þ: 609.1933; Found: 609.1872.
6: 1H NMR (500MHz, CDCl3): d 0.00 (s, 9H, SiMe3), 0.95 (m, 2H, H-20), 1.15, 1.29 (2s,
18H, 2CMe3), 2.57(m, 1H, OH), 3.47(m, 1H, H-6a), 3.72(m, 1H, H-6b), 3.59(m, 1H, H-10a),
3.78 (m, 1H, H-5), 3.97 (m, 1H, H-10b), 4.22 (m, 1H, H-2), 4.69 (d, J ¼ 8.3 Hz, 1H, H-1),
5.29–5.38 (m, 2H, H-3, H-4), 6.75 (m, 1H, NH); 13C NMR (125MHz, CDCl3): d 21.5
(SiMe3), 18.3 (C-20), 27.0, 27.5 (2CMe3), 38.9, 39.2 (2CMe3), 53.2 (C-2), 60.4 (C-6), 67.1 (C-
10), 67.8 (C-4), 69.9 (C-3), 73.8 (C-5), 92.4 (CCl3), 100.6 (C-1), 161.8, 178.2, 178.5 (3CO).
2-(Trimethylsilyl)ethyl 2-Deoxy-2-trichloroacetamido-b-D-
galactopyranoside (7)
Compound 5 (2.49 g, 4.19 mmol) was treated with 1 M NaOMe/MeOH (2 mL) in
methanol (20 mL) at rt. After 7 hr, the mixture was neutralized with Dowex 50 X 8 (Hþ)
ion-exchange resin and filtered through a pad of Celite. The Celite was washed with

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14
Schwardt et al.
methanol (3 ꢀ 5 mL) and the combined filtrates were concentrated in vacuo to give
galactoside 7 (1.71 g, 96%) as a colorless solid.
[a]D 23.3 (c ¼ 0.59, CH3OH); 1H NMR (500 MHz, CD3OD): d 0.00 (s, 9H, SiMe3),
0.92 (m, 2H, H-20), 3.51 (ddd, J ¼ 1.2, 5.4, 6.8 Hz, 1H, H-5), 3.58 (ddd, J ¼ 6.2, 9.6,
10.7 Hz, 1H, H-10a), 3.74 (dd, J ¼ 5.4, 11.3 Hz, 1H, H-6a), 3.77 (dd, J ¼ 3.2, 10.9 Hz, 1H,
H-3), 3.78 (dd, J ¼ 6.8, 11.3 Hz, 1H, H-6b), 3.85 (dd, J ¼ 1.3, 3.5 Hz, 1H, H-4), 3.96 (ddd,
J ¼ 8.5, 9.0, 11.0 Hz, 1H, H-2), 3.99 (ddd, J ¼ 6.0, 9.5, 10.7 Hz, 1H, H-10b), 4.54
(d, J ¼ 8.4 Hz, 1H, H-1), 8.62 (d, J ¼ 9.0 Hz, 1H, NH); 13C NMR (125 MHz, CD3OD):
d 21.5 (SiMe3), 19.0 (C-20), 56.2 (C-2), 62.5 (C-6), 67.8 (C-10), 69.6 (C-4), 72.1 (C-3),
76.5 (C-5), 94.1 (CCl3), 101.9 (C-1), 164.3 (CO); HRMS (FAB) Calcd for C13H24Cl3-
NO6Si [M 2 NH4]þ: 441.0782; Found: 441.0780.
2-(Trimethylsilyl)ethyl 4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-
b-D-galactopyranoside (8)
To a solution of 7 (1.69 g, 3.98 mmol) and benzaldehyde dimethyl acetal (895 mL,
5.97 mmol) in acetonitrile (30 mL) was added p-toluene sulfonic acid monohydrate
(75.7 mg, 0.398 mmol) at rt. The mixture was stirred for 4 hr before NEt3 (2 mL) was
added. The solvent was removed, and the residue was purified by column chromatography
on silica gel (petroleum ether/EtOAc, gradient 1 : 1 to 1 : 2) to yield product 8 (1.75 g,
86%) as a colorless foam.
1
[a]D 27.2 (c ¼ 1.00, CHCl3); H NMR (500 MHz, CDCl3): d 0.00 (s, 9H, SiMe3),
0.96 (m, 2H, H-20), 2.76 (m, 1H, OH), 3.54 (m, 1H, H-5), 3.56 (m, 1H, H-10a), 3.74 (ddd,
J ¼ 7.1, 8.3, 10.1 Hz, 1H, H-2), 4.03 (m, 1H, H-10b), 4.10 (dd, J ¼ 1.9, 12.5 Hz, 1H,
H-6a), 4.25 (m, 2H, H-3, H-4), 4.36 (dd, J ¼ 1.6, 12.5 Hz, 1H, H-6b), 4.84 (d, J ¼ 8.3 Hz,
1H, H-1), 5.59 (s, 1H, PhCH), 6.87 (d, J ¼ 7.1 Hz, 1H, NH), 7.36–7.38, 7.50–7.52 (m, 5H,
C6H5); 13C NMR (125 MHz, CDCl3): d 21.4 (SiMe3), 18.1 (C-20), 57.0 (C-2), 66.6 (C-5),
67.3 (C-10), 69.1 (C-3), 69.1 (C-6), 75.0 (C-4), 92.5 (CCl3), 99.0 (C-1), 101.3 (PhCH),
126.4, 128.2, 129.3, 137.3 (C6H5), 162.4 (CO); HRMS (FAB) Calcd for C20H28Cl3NO6Si
[M 2 NH4]þ: 529.1095; Found: 529.1078.
2-(Trimethylsilyl)ethyl (2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!3)-4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-b-D-
galactopyranoside (10)
A solution of acceptor 8 (500 mg, 0.975 mmol) and ethyl 1-thio-D-galactopyranoside
9[33] (920 mg, 1.44 mmol) in DCM (10 mL) containing 3 A molecular sieves (2 g) was
˚
stirred at 2258C under argon. After 1.5 hr, N-iodosuccinimide (NIS; 660 mg, 2.93 mmol)
was added, and stirring was continued for 30 min. Trifluoromethane sulfonic acid (TfOH;
25.5 mL, 0.293 mmol) in DCM (200 mL) was then added dropwise at 2258C. The reaction
mixture was stirred for additional 1.5 hr at 2258C, diluted with DCM (20 mL), and filtered
through a pad of Celite. The Celite was washed with DCM, and the combined filtrates were
extracted with 10% aqueous Na2S2O3 (10 mL) and saturated aqueous KHCO3 (10 mL).
The organic layer was dried (Na2SO4), filtered and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel (petroleum ether/

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Recombinant Rat ST3Gal III
15
EtOAc, gradient 5 : 1 to 2 : 1) to yield the b-disaccharide 10 (998 mg, 94%) as a 4 : 1
mixture with its a-anomer. After a final crystallization from (i-Pr)2O, the pure b-anomer
10 was isolated as a colorless solid (655 mg, 64%).
[a]D þ77.6 (c ¼ 0.50, CHCl3); 1H NMR (500 MHz, CDCl3): d 20.03 (s, 9H, SiMe3),
0.89 (m, 2H, H-200), 3.25 (m, 1H, H-5), 3.51 (m, 1H, H-100a), 3.67 (ddd, J ¼ 6.5, 8.2,
11.2 Hz, 1H, H-2), 3.82 (dd, J ¼ 1.8, 12.3 Hz, 1H, H-6a), 3.95 (m, 1H, H-100b), 4.23 (dd,
J ¼ 1.6, 12.3 Hz, 1H, H-6b), 4.35 (ddd, J ¼ 1.1, 4.8, 7.6 Hz, 1H, H-50), 4.41 (m, 1H, H-4),
4.43 (dd, J ¼ 4.8, 11.6 Hz, 1H, H-60a), 4.73 (dd, J ¼ 7.6, 11.6 Hz, 1H, H-60b), 4.78 (dd,
J ¼ 3.6, 11.2 Hz, 1H, H-3), 5.09 (d, J ¼ 8.2 Hz, 1H, H-1), 5.28 (d, J ¼ 7.9 Hz, 1H, H-10),
5.51 (s, 1H, PhCH), 5.57 (dd, J ¼ 3.4, 10.4 Hz, 1H, H-30), 5.87 (dd, J ¼ 7.9, 10.4 Hz, 1H,
H-20), 5.96 (dd, J ¼ 1.1, 3.4 Hz, 1H, H-40), 7.02 (d, J ¼ 6.4 Hz, 1H, NH), 7.30–7.34,
7.37–7.49, 7.56–7.61, 7.73–7.76, 7.89, 8.04–8.05 (m, 25H, 5C6H5); 13C NMR
(125 MHz, CDCl3): d 21.5 (SiMe3), 18.1 (C-200), 55.6 (C-2), 62.7 (C-60), 66.5 (C-5), 67.4
(C-100), 68.2 (C-40), 68.9 (C-6), 69.8 (C-20), 71.6 (C-50), 71.8 (C-30), 74.1 (C-3), 76.2 (C-4),
92.1 (CCl3), 97.6 (C-1), 100.5 (C-10), 101.2 (PhCH), 126.1, 128.1, 128.2, 128.4, 128.5,
128.6, 128.6, 128.8, 128.8, 129.1, 129.3, 129.7, 129.7, 129.8, 129.9, 133.3, 133.4, 133.6,
137.8 (5C6H5), 162.1, 165.1, 165.2, 165.9 (5CO); HRMS (FAB) Calcd for C54H54Cl3-
NO15Si [M-NH4]þ: 1107.2672; Found: 1107.2655.
2-(Trimethylsilyl)ethyl b-D-Galactopyranosyl-(1!3)-2-Deoxy-2-
trichloroacetamido-b-D-galactopyranoside (11)
Compound 10 (48.5 mg, 44.5mmol) was treated with 1 M NaOMe/MeOH (0.5 mL) in
methanol (5mL) at rt. After 18hr, the mixture was neutralized with Dowex 50 X 8 (Hþ) ion-
exchange resin and filtered through a pad of Celite. The Celite was washed with methanol
(3 ꢀ 3 mL), and the combined filtrates were concentrated at reduced pressure. The residue
was dissolved in 80% aqueous acetic acid (3 mL) and stirred for 2 hr at 808C. The solvent was
evaporated in vacuo, and the remaining solid was purified by column chromatography on
silica gel (DCM/MeOH 10: 1) to give disaccharide 11 (12.1 mg, 46%) as a colorless solid.
[a]D 23.4 (c ¼ 0.25, CH3OH); 1H NMR (500 MHz, CD3OD): d 0.00 (s, 9H, SiMe3),
0.92 (m, 2H, H-200), 3.42 (dd, J ¼ 2.8, 9.7 Hz, 1H, H-30), 3.49 (m, 1H, H-50), 3.53–3.56
(m, 2H, H-5, H-20), 3.59 (m, 1H, H-100a), 3.69 (dd, J ¼ 4.8, 11.4 Hz, 1H, H-60a), 3.73–3.78
(m, 3H, H-6, H-60b), 3.80 (m, 1H, H-40), 3.99 (m, 1H, H-100b), 4.00–4.01 (m, 2H, H-2,
H-3), 4.13 (m, 1H, H-4), 4.38 (d, J ¼ 7.6 Hz, 1H, H-10), 4.65 (d, J ¼ 8.0 Hz, 1H, H-1); 13C
NMR (125 MHz, CD3OD): d 21.3 (SiMe3), 17.9 (C-200), 55.0 (C-2), 61.3 (C-6), 61.4
(C-60), 66.9 (C-100), 68.3 (C-4), 69.2 (C-40), 71.7 (C-30), 71.8 (C-20), 74.9 (C-5), 75.2 (C-
50), 78.7 (C-3), 101.4 (C-1), 105.1 (C-10); HRMS (FAB) Calcd for C19H34Cl3NO11Si
[M 2 NH4]þ: 603.1310; Found: 603.1280.
2-(Trimethylsilyl)ethyl (2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!3)-2-Acetamido-4,6-O-benzylidene-2-deoxy-b-D-
galactopyranoside (12)
A solution of 10 (60.0 mg, 55.0 mmol), tributyltin hydride (58.0 mL, 220 mmol), and
AIBN (2 mg) in dry benzene (2 mL) was stirred for 45 min at rt under argon, then heated
under reflux for 2.5 hr, cooled, and concentrated under reduced pressure. The remains were

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16
Schwardt et al.
dissolved in acetonitrile (10 mL) and washed with hexane (5 mL). The acetonitrile was
evaporated, and the residue was precipitated from EtOAc/hexane to give 12 (50.4 mg,
93%) as a colorless solid.
[a]D þ80.1 (c ¼ 0.50, CHCl3); 1H NMR (500 MHz, CDCl3): d 20.04 (s, 9H,
SiMe3), 0.80–0.91 (m, 2H, H-200), 1.34 (s, 3H, CH3), 3.26–3.31 (m, 2H, H-2, H-5),
3.45 (m, 1H, H-100a), 3.67 (A of AB, J ¼ 12.1 Hz, 1H, H-6A), 3.94 (m, 1H, H-100b),
4.17 (B of AB, J ¼ 12.1 Hz, 1H, H-6B), 4.35–4.41 (m, 3H, H-4, H-50, H-60a), 4.74
(dd, J ¼ 6.6, 10.6 Hz, 1H, H-60b), 4.82 (dd, J ¼ 3.5, 11.0 Hz, 1H, H-3), 5.07 (m, 2H,
H-1, H-10), 5.41 (d, J ¼ 6.6 Hz, 1H, NH), 5.46 (s, 1H, PhCH), 5.61 (dd, J ¼ 3.3,
10.3 Hz, 1H, H-30), 5.84 (dd, J ¼ 8.0, 10.2 Hz, 1H, H-20), 5.96 (m, 1H, H-40), 7.21–
7.24, 7.34–7.62, 7.76–7.77, 7.94–7.95, 8.02–8.07 (m, 25H, 5C6H5); 13C NMR
(125 MHz, CDCl3): d 21.4 (SiMe3), 17.9 (C-200), 23.1 (CH3), 54.9 (C-2), 62.7 (C-60),
66.4 (C-5), 66.8 (C-100), 68.2 (C-40), 69.1 (C-6), 69.9 (C-20), 71.4 (C-50), 71.7 (C-30),
76.0 (C-4), 76.2 (C-3), 98.0 (C-1), 100.7 (C-10), 102.5 (PhCH), 126.3, 128.1, 128.3,
128.5, 128.6, 128.7, 128.9, 129.2, 129.7, 129.7, 130.0, 133.3, 133.4, 133.5, 133.6,
138.0 (5C6H5), 164.9, 165.9, 166.0, 171.0 (5CO); HRMS (FAB) Calcd for
C54H57NO15Si [M 2 NH4]þ: 1005.3841; Found: 1005.3825.
2-(Trimethylsilyl)ethyl b-D-Galactopyranosyl-(1!3)-2-Acetamido-2-
deoxy-b-D-galactopyranoside (13)
A solution of 12 (50.0 mg, 50.6 mmol) in 80% aqueous acetic acid (3 mL) was stirred
for 4.5 hr at 508C. The solvent was evaporated in vacuo, and the remaining solid was
treated with 1 M NaOMe/MeOH (0.4 mL) in methanol (4 mL) at rt. After 21 hr, the
mixture was neutralized with Dowex 50 X 8 (Hþ) ion-exchange resin and filtered through
a pad of Celite. The Celite was washed with methanol (3 ꢀ 3 mL), and the combined
filtrates were concentrated at reduced pressure. The residue was purified by column
chromatography on RP-18 (H2O/MeOH, gradient 1 : 0 to 1 : 1) to give disaccharide 13
(15.7 mg, 64%) as a colorless solid.
[a]D 27.8 (c ¼ 0.25, CH3OH); 1H NMR (500 MHz, CD3OD): d 0.02 (s, 9H, SiMe3),
0.86 (ddd, J ¼ 5.4, 9.7, 14.0 Hz, 1H, H-200a), 0.96 (ddd, J ¼ 7.0, 10.2, 14.0 Hz, 1H,
H-200b), 1.95 (s, 3H, CH3), 3.45 (dd, J ¼ 3.3, 9.7 Hz, 1H, H-30), 3.48–3.58 (m, 4H, H-5,
H-20, H-50, H-100a), 3.71 (dd, J ¼ 5.1, 11.4 Hz, 1H, H-60a), 3.72–3.78 (m, 4H, H-3, H-6,
H-60b), 3.82 (m, 1H, H-40), 3.99 (dd, J ¼ 8.5, 10.5 Hz, 1H, H-2), 4.03 (m, 1H, H-100b), 4.10
(m, 1H, H-4), 4.31 (d, J ¼ 7.6 Hz, 1H, H-10), 4.49 (d, J ¼ 8.5 Hz, 1H, H-1); 13C NMR
(125 MHz, CD3OD): d 21.3 (SiMe3), 18.8 (C-200), 23.3 (CH3), 53.1 (C-2), 62.5, 62.6 (C-6,
C-60), 67.7 (C-100), 69.7 (C-4), 70.3 (C-40), 72.5 (C-30), 74.6 (C-20), 76.3, 76.8 (C-5, C-50),
81.9 (C-3), 102.2 (C-1), 106.7 (C-10), 174.2 (CO); HRMS (FAB) Calcd for C19H37NO11Si
[M 2 H]2: 484.2214; Found: 484.2219.
2-(Trimethylsilyl)ethyl (2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!3)-2,6-di-O-Benzyl-b-D-galactopyranoside (15)
A
mixture of 2-(trimethylsilyl)ethyl 2,6-di-O-benzyl-b-D-galactopyranoside
(14,[17,18] 300 mg, 0.651 mmol), ethyl 1-thio-D-galactopyranoside 9[33] (500 mg,

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Recombinant Rat ST3Gal III
17
˚
0.780 mmol) and activated powdered molecular sieves 3 A (2.0 g) in DCM was stirred
for 6 hr at rt under argon. At 08C DMTST (504 mg, 1.95 mmol) was then added in one
portion. The reaction mixture was stirred at 78C for additional 16 hr, diluted with DCM
(10 mL), and filtered through a pad of Celite. The Celite was washed with DCM
(3 ꢀ 10 mL), and the combined filtrates were washed with saturated aqueous KHCO3
(2 ꢀ 20 mL) and H2O (20 mL). The organic layer was dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by silica gel
chromatography (petroleum ether/EtOAc 5 : 1) to afford disaccharide 15 (588 mg,
87%) as a colorless foam.
1
[a]D þ56.4 (c ¼ 1.00, CHCl3); H NMR (500MHz, CDCl3): d 0.00 (s, 9H, SiMe3),
0.95 (m, 2H, H-200), 3.54 (ddd, J ¼ 5.8, 9.6, 11.5Hz, 1H, H-100a), 3.58–3.62 (m, 2H, H-2,
H-5), 3.71 (dd, J ¼ 5.1, 10.1Hz, 1H, H-6a), 3.75 (dd, J ¼ 6.6, 10.1Hz, 1H, H-6b), 3.84 (dd,
J ¼ 3.3, 9.4 Hz, 1H, H-3), 4.00 (ddd, J ¼ 6.1, 9.6, 11.4Hz, 1H, H-100b), 4.13 (d, J ¼ 2.8 Hz,
1H, H-4), 4.28 (m, 1H, H-50), 4.34 (d, J ¼ 7.8 Hz, 1H, H-1), 4.36 (A of AB, J ¼ 11.1Hz,
1H, PhCH2), 4.45 (dd, J ¼ 6.1, 11.4Hz, 1H, H-60a), 4.55 and 4.59 (A, B of AB, J ¼ 11.9Hz,
2H, PhCH2), 4.65 (dd, J ¼ 7.0, 11.4Hz, 1H, H-60b), 4.72 (B of AB, J ¼ 11.0Hz, 1H,
PhCH2), 5.27 (d, J ¼ 8.0 Hz, 1H, H-10), 5.62 (dd, J ¼ 3.5, 10.5Hz, 1H, H-30), 5.89 (dd,
J ¼ 8.0, 10.5Hz, 1H, H-20), 6.00 (dd, J ¼ 0.8, 3.5 Hz, 1H, H-40), 7.16–7.18, 7.25–7.32,
7.35–7.37, 7.43–7.47, 7.53–7.68, 7.78–7.80, 7.87–7.89, 8.03–8.05, 8.12–8.14 (m, 30H,
6C6H5); 13C NMR (125MHz, CDCl3): d 21.5 (SiMe3), 18.4 (C-200), 62.0 (C-60), 67.3 (C-
100), 68.1 (C-40), 68.9 (C-4), 69.5 (C-6), 69.9 (C-20), 71.4, 71.5 (C-30, C-50), 73.2 (C-5), 73.6
(PhCH2), 74.7 (PhCH2), 78.9 (C-2), 80.8 (C-3), 101.6 (C-10), 102.9 (C-1), 127.5–138.5
(36C, 6C6H5), 165.5, 165.5, 165.6, 165.9 (4CO); HRMS (FAB) Calcd for C59H62O15Si
[M 2 NH4]þ: 1056.4202; Found: 1056.4198.
2-(Trimethylsilyl)ethyl b-D-Galactopyranosyl-(1!3)-b-D-
Galactopyranoside (16)
To a solution of 15 (83.0 mg, 79.9 mmol) in methanol (5 mL) was added 1 M NaOMe/
MeOH (0.5 mL). The reaction mixture was stirred at rt under argon for 2 hr, then
neutralized with Amberlite IRC 50 ion-exchange resin, filtered and concentrated under
reduced pressure. The residue was dissolved in MeOH (2.5 mL) containing a catalytic
amount of acetic acid and hydrogenolyzed (1 bar H2) in the presence of 10% Pd–C
(15 mg) for 3 hr at rt. The reaction mixture was filtered through a pad of Celite, which was
washed with MeOH (5 mL). The combined filtrates were concentrated, and the residue was
purified by silica gel chromatography (DCM/MeOH 3 : 1) to afford disaccharide 16
(26.6 mg, 75%) as a colorless foam.
[a]D22.3 (c ¼ 1.00, MeOH); 1H NMR (500MHz, CD3OD): d 0.00 (s, 9H, SiMe3), 0.94
(m, 1H, H-200a), 1.02 (m, 1H, H-200b), 3.46 (dd, J ¼ 3.2, 9.7 Hz, 1H, H-30), 3.49–3.52 (m, 2H,
H-5, H-50), 3.55–3.74 (m, 8H, H-2, H-3, H-6, H-20, H-60, H-100a), 3.75 (d, J ¼ 3.2 Hz, 1H,
H-40), 3.98 (ddd, J ¼ 5.7, 9.6, 11.5Hz, 1H, H-100b), 4.08 (d, J ¼ 3.0 Hz, 1H, H-4), 4.26
(d, J ¼ 7.7 Hz, 1H, H-1), 4.45 (d, J ¼ 7.6 Hz, 1H, H-10); 13C NMR (125MHz, CD3OD):
d 21.5 (SiMe3), 19.0 (C-200), 62.4 (2C, C-6, C-60), 67.9 (H-100), 69.7, 70.1 (C-4, C-40), 71.4,
72.9 (C-2, C-20), 74.5, 76.1 (C-5, C-50), 76.7 (C-30), 85.0 (C-3), 103.8 (C-1), 106.3 (C-10);
HRMS (FAB) Calcd for C17H34O11Si [M-Na]þ: 465.1768; Found: 465.1762.

ORDER
REPRINTS
18
Schwardt et al.
2-(Trimethylsilyl)ethyl (2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!3)-b-D-Galactopyranoside (17)
Compound 15 (100 mg, 96.2 mmol) was dissolved in methanol (2.0 mL) and
dioxane (1.0 mL) containing a trace of acetic acid and hydrogenolyzed (4 bar H2) in the
presence of 10% Pd–C (150 mg) for 9 d. The reaction mixture was filtered through a
pad of Celite, which was washed with DCM (5 mL). The combined filtrates were
concentrated, and the residue was purified by silica gel chromatography (petroleum
ether/EtOAc 2 : 1) to afford 17 (55.4 mg, 67%) as colorless oil, which was immediately
used in the next step.
1H NMR (500 MHz, CDCl3): d 0.00 (s, 9H, SiMe3), 0.97 (m, 2H, H-200), 3.47
(t, J ¼ 5.6 Hz, 1H, H-5), 3.54 (m, 1H, H-100a), 3.65–3.68 (m, 2H, H-3, H-6a), 3.73 (m, 1H,
H-2), 3.89 (dd, J ¼ 6.6, 11.7 Hz, 1H, H-6b), 3.99 (m, 1H, H-100b), 4.08 (d, J ¼ 2.9 Hz, 1H,
H-4), 4.22 (d, J ¼ 7.6 Hz, 1H, H-1), 4.39 (m, 1H, H-50), 4.48 (dd, J ¼ 5.6, 11.5 Hz, 1H,
H-60a), 4.68 (dd, J ¼ 7.2, 11.5 Hz, 1H, H-60b), 5.16 (d, J ¼ 7.9 Hz, 1H, H-10), 5.68 (dd,
J ¼ 3.5, 10.5 Hz, 1H, H-30), 5.81 (dd, J ¼ 7.9, 10.4 Hz, 1H, H-20), 6.01 (d, J ¼ 3.2 Hz, 1H,
H-40), 7.26–7.29, 7.37–7.40, 7.45–7.48, 7.51–7.55, 7.59–7.62, 7.65–7.68, 7.80–7.81,
7.97–7.98, 8.03–8.05, 8.12–8.13 (m, 20H, 4C6H5); MS (ESI) Calcd for C45H50O15Si
[M þ Na]þ: 881.6; Found: 881.6.
2-(Trimethylsilyl)ethyl (2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!3)-[(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosynate)]-(2!6)-b-D-
Galactopyranoside (19)
To a solution of 17 (50.0 mg, 58.2 mmol) and methyl(methyl 5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-D-galacto-2-nonulopyranosid)onate (18,[37]
60.7 mg, 0.116 mmol) in MeCN/DCM (3 : 1, 4 mL) was added activated powdered
˚
molecular sieves 3 A (1.0 g). The reaction mixture was stirred at rt under argon for 6 hr and
then cooled to 2258C. After the addition of NIS (52.4 mg, 0.23 mmol) and TfOH (2.0 mL,
0.02 mmol), stirring was continued for 16 hr at 2258C. Then the mixture was diluted with
DCM (10 mL) and filtered through a pad of Celite. The Celite was washed with DCM
(3 ꢀ 10 mL), and the combined filtrates were extensively washed with 20% aqueous
Na2S2O3 (30 mL), saturated aqueous KHCO3 (2 ꢀ 20mL) and H2O (20 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by
silica gel chromatography (DCM/MeOH 60 : 1) to afford the trisaccharide 19 (35.0 mg,
45%) as a colorless foam.
1H NMR (500 MHz, CDCl3): d 0.00 (s, 9H, SiMe3), 0.98 (m, 2H, H-2000), 1.89, 2.02,
2.03, 2.12, 2.15 (5s, 15H, 5CH3), 1.94 (m, 1H, H-300a), 2.59 (dd, J ¼ 4.6, 12.8 Hz, 1H,
H-300b), 3.51–3.58 (m, 2H, H-5, H-1000a), 3.65–3.68 (m, 2H, H-2, H-6a), 3.74 (dd, J ¼ 3.2,
9.4 Hz, 1H, H-3), 3.81 (s, 3H, OCH3), 3.88 (dd, J ¼ 5.5, 9.6 Hz, 1H, H-6b), 3.98 (m, 1H,
H-1000), 4.06–4.13 (m, 4H, H-4, H-500, H-600, H-900a), 4.19 (d, J ¼ 7.7 Hz, 1H, H-1), 4.30–
4.38 (m, 2H, H-900b, H-50), 4.42 (dd, J ¼ 7.0, 11.1 Hz, 1H, H-60a), 4.70 (dd, J ¼ 6.2,
11.1 Hz, 1H, H-60b), 4.86 (ddd, J ¼ 4.6, 9.8, 12.2 Hz, 1H, H-400), 5.24 (d, J ¼ 9.6 Hz, 1H,
NH), 5.30–5.34 (m, 2H, H-10, H-700), 5.36–5.40 (m, 1H, H-800), 5.66 (dd, J ¼ 3.5, 10.4 Hz,
1H, H-30), 5.80 (dd, J ¼ 8.0, 10.4 Hz, 1H, H-20), 6.02 (d, J ¼ 3.4 Hz, 1H, H-40), 7.24–7.27,

ORDER
REPRINTS
Recombinant Rat ST3Gal III
19
7.36–7.39, 7.43–7.47, 7.50–7.55, 7.57–7.60, 7.63–7.66, 7.78–7.80, 7.95–7.97,
8.01–8.03, 8.10–8.12 (m, 20H, 4C6H5); 13C NMR (125 MHz, CDCl3): d 21.4 (SiMe3),
18.1 (C-2000), 20.8, 20.8, 21.0, 23.2 (5CH3), 37.6 (C-300), 49.4 (C-500), 52.9 (OCH3), 61.7 (C-
60), 62.4 (C-900), 62.6 (C-6), 67.1 (C-1000), 67.5 (2C, C-4, C-700), 67.6 (C-40), 68.0 (2C, C-400,
C-800), 68.9 (C-20), 70.0 (C-2), 71.1, 71.4 (C-30, C-50), 72.3 (C-600), 72.8 (C-5), 81.2 (C-3),
99.0 (C-200), 101.9, 102.1 (C-1, C-10), 128.3, 128.4, 128.5, 128.6, 128.7, 129.0, 129.2,
129.3, 129.7, 129.8, 130.0, 131.0, 133.3, 133.4, 133.6 (24C, 4C6H5), 165.5, 165.6, 165.6,
169.0, 169.9, 170.1, 170.3, 170.7, 170.9 (10C, 10CO); HRMS (FAB) Calcd for
C65H77NO27Si [M 2 NH4]þ: 1349.4796; Found: 1349.4778.
2-(Trimethylsilyl)ethyl b-D-Galactopyranosyl-(1!3)-[Sodium
(5-Acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosynate)]-(2!6)-b-D-Galactopyranoside (20)
To a solution of 19 (32.5 mg, 24.4 mmol) in methanol (1 mL) was added freshly
prepared 1 M NaOMe/MeOH (1.0 mL). The mixture was stirred at rt under argon for 7 hr,
then water (1 mL) was added and the mixture was stirred for another 16 hr at rt. The
solution was concentrated, and the residue was purified by reverse phase chromatography
(RP-18 column, 5% gradient MeOH in water), Dowex ion-exchange chromatography
(Naþ type), and P2 size exclusion chromatography to afford 20 (17.8 mg, 90%) as a
colorless solid after a final lyophilization from water.
1
[a]D 23.5 (c ¼ 1.00, H2O); H NMR (500MHz, D2O): d 0.00 (s, 9H, SiMe3), 0.95
(ddd, J ¼ 5.2, 12.9Hz, 1H, H-2000a), 1.04 (ddd, J ¼ 5.5, 12.9Hz, 1H, H-2000b), 1.63
(t, J ¼ 12.2Hz, 1H, H-300a), 2.00 (s, 3H, CH3), 2.68 (dd, J ¼ 4.7, 12.4Hz, 1H, H-300b), 3.55
(m, 1H, H-600), 3.56–3.60 (m, 3H, H-2, H-20, H-900a), 3.61–3.64 (m, 2H, H-6a, H-30), 3.66–
3.68 (m, 3H, H-50, H-400, H-700), 3.71 (m, 1H, H-60a), 3.72–3.79 (m, 4H, H-3, H-60b, H-800,
H-1000a), 3.81–3.85 (m, 3H, H-5, H-6b, H-500), 3.87–3.91 (m, 2H, H-40, H-900b), 4.00 (ddd,
J ¼ 5.1, 10.1, 12.7Hz, H-1000b), 4.20 (d, J ¼ 3.3 Hz, 1H, H-4), 4.42 (d, J ¼ 8.0 Hz, 1H, H-1),
13
4.55 (d, J ¼ 7.6 Hz, 1H, H-10); C NMR (125MHz, D2O): d 22.2 (SiMe3), 17.1 (C-2000),
39.900(0C-300), 52.2 (C-500), 61.4 (C-60), 63.0 (C-6), 63.4 (C-900), 68.5, 68.5 (C-50, C-600), 68.5
(C-1 ), 68.9 (2C, C-4, C-40), 70.0 (C-2), 71.3 (2C, C-20, C-700), 72.0 (C-5), 72.9 (C-400), 73.3
(C-800), 75.4 (C-30), 83.0 (C-3), 100.2 (C-200), 102.1 (C-1), 105.0 (C-10), 173.2, 175.4 (CO);
MS (ESI) Calcd for C28H50NNaO19Si [M þ Na]þ: 778.3; Found: 778.5 (100%).
8-(Methoxycarbonyl)octyl (5-Acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosylonic acid)-(2!3)-b-D-Galactopyranosyl-
(1!4)-2-Acetamido-2-deoxy-b-D-glucopyranoside (23)
Disaccharide 21[40] (3.0 mg, 5.4 mmol) and CMP-NeuAc (5.3 mg, 8.1 mmol) were
dissolved in a mixture of 50 mM sodium cacodylate buffer pH 6.5 (0.6 mL), 60 mM
aqueous MnCl2 (0.6 mL) and deionized water (0.4 mL) containing BSA (0.4 mg, Fluka).
The mixture was incubated at 378C with CIAP (2 mL) and recombinant ST3Gal III
(200 mL, 9 U/L). After 3 d TLC (silica gel, DCM/MeOH/H2O 10 : 4 : 0.8) indicated
complete consumption of 21. The turbid solution was centrifuged, and the supernatant was
passed over a RP-18 column, which was washed with water before the product was eluted

ORDER
REPRINTS
20
Schwardt et al.
with MeOH. After evaporation of methanol, the residue was chromatographed on silica gel
(DCM/MeOH/H2O 10 : 4 : 0.4, then 6 : 4 : 1) to yield trisaccharide 23 (3.5 mg, 76%) as
colorless powder after a final lyophilization from water. The spectroscopic data of 23 were
in accordance to those reported.[20a,29a]
8-(Methoxycarbonyl)octyl (5-Acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosylonic acid)-(2!3)-b-D-Galactopyranosyl-
(1!3)-2-Acetamido-2-deoxy-b-D-glucopyranoside (24)
According to the procedure described for the synthesis of 23, compound 22[41]
(3.0 mg, 5.4 mmol) was incubated with CMP-NeuAc (5.3 mg, 8.1 mmol) and ST3Gal III
(150 mL, 9 U/L). Reaction control by TLC (silica gel, DCM/MeOH/H2O 10 : 4 : 0.8)
indicated complete consumption of 22 after 3 d. After work-up, the crude product was
purified on RP-18 (H2O/MeOH gradient 1 : 0 to 1 : 1) to yield trisaccharide 24 (4.1 mg,
90%) as colorless powder after a final lyophilization from water. The spectroscopic data of
24 were in accordance to those reported.[29b,42]
2-(Trimethylsilyl)ethyl (5-Acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
2-nonulopyranosylonic acid)-(2!3)-b-D-Galactopyranosyl-(1!3)-2-
Acetamido-2-deoxy-b-D-galactopyranoside (25)
Disaccharide 13 (10.0 mg, 20.6 mmol) and CMP-NeuAc (20.4 mg, 31.1 mmol) were
dissolved in a mixture of 50 mM sodium cacodylate-buffer pH 6.5 (2 mL), 60 mM
aqueous MnCl2 (2 mL) and deionized water (1.3 mL) containing BSA (2.0 mg). The
mixture was incubated at 378C with CIAP (2 mL) and recombinant ST3Gal III (500 mL,
9 U/L). After 17 hr, additional ST3Gal III (250 mL) was added, and the incubation was
continued for 4 d. The turbid solution was centrifuged, and the supernatant was passed
over a RP-18 column, which was washed with water before the crude product was
eluted with MeOH. After evaporation of the solvent, the residue was chromatographed
on RP-18 (H2O/MeOH gradient 1 : 0 to 1 : 1) to yield trisaccharide 25 (9.0 mg, 56%)
and starting material 13 (4.4 mg, 44%) as colorless powders after a final lyophilization
from water.
1
[a]D 213.1 (c ¼ 0.25, H2O); H NMR (500 MHz, D2O): d 0.00 (s, 9H, SiMe3),
0.86 (m, 1H, H-2000a), 0.97 (m, 1H, H-2000b), 1.78 (t, J ¼ 11.6 Hz, 1H, H-300a), 2.00, 2.02
(s, 6H, 2CH3), 2.74 (m, 1H, H-300b), 3.54 (m, 1H, H-20), 3.57–3.62 (m, 3H, H-5, H-600,
H-700), 3.63–3.74 (m, 6H, H-6, H-60a, H-400, H-900a, H-1000a), 3.75–3.81 (m, 2H, H-50,
H-60b), 3.82–3.87 (m, 4H, H-3, H-500, H-800, H-900b), 3.92 (m, 1H, H-40), 3.97–4.06
(m, 3H, H-2, H-30, H-1000b), 4.16 (m, 1H, H-4), 4.49 (d, J ¼ 7.6 Hz, 1H, H-10), 4.53
13
(d, J ¼ 8.3 Hz, 1H, H-1); C NMR (125 MHz, D2O): d 21.8 (SiMe3), 17.6 (C-2000),
22.4, 22.6 (2CH3), 40.1 (C-300), 51.6 (C-2), 52.2 (C-500), 61.3, 61.4 (C-6, C-60), 62.7 (C-
900), 67.7 (C-40), 68.2 (C-4), 68.3 (C-700), 68.5 (C-400), 68.6 (C-1000), 69.2 (C-20), 72.2 (C-
800), 72.7 (C-600), 75.0, 75.1 (C-5, C-50), 76.1 (C-30), 80.8 (C-3), 100.2 (C-100), 100.8 (C-
1), 104.9 (C-10); HRMS (FAB) Calcd for C30H54N2O19Si [M 2 H]2: 775.3168; Found:
775.3110.

ORDER
REPRINTS
Recombinant Rat ST3Gal III
21
2-(Trimethylsilyl)ethyl (5-Acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
2-nonulopyranosylonic acid)-(2!3)-b-D-Galactopyranosyl-(1!3)-2-
deoxy-2-trichloroacetamido-b-D-galactopyranoside (26)
According to the procedure described for the synthesis of 25, compound 11 (12.0 mg,
20.4 mmol) was incubated with CMP-NeuAc (20.2 mg, 30.1 mmol), BSA (2.1 mg), CIAP
(2 mL) and ST3Gal III (500 mL, 9 U/L). After 21 hr additional ST3Gal III (250 mL) was
added, and the incubation was continued for 3 d. After work-up, the residue was
chromatographed on silica gel (DCM/MeOH/H2O 10 : 4 : 0.4, then 6 : 4 : 1) to yield
starting material 11 (7.5 mg, 62%) and trisaccharide 26 (6.4 mg, 36%) as colorless
powders after a final lyophilization from water.
1
[a]D 28.5 (c ¼ 0.25, H2O); H NMR (500 MHz, D2O): d 20.03 (s, 9H, SiMe3),
0.89 (m, 1H, H-2000a), 0.98 (m, 1H, H-2000b), 1.75 (t, J ¼ 12.2 Hz, 1H, H-300a), 2.00
(s, 3H, CH3), 2.72 (dd, J ¼ 4.6, 12.4 Hz, 1H, H-300b), 3.52 (dd, J ¼ 7.8, 9.7 Hz, 1H,
H-20), 3.56 (dd, J ¼ 1.8, 10.5 Hz, 1H, H-600), 3.59–3.61 (m, 3H, H-5, H-6a, H-700),
3.62–3.74 (m, 6H, H-6b, H-50, H-60a, H-400, H-900a, H-1000a), 3.75–3.83 (m, 3H, H-60b,
H-500, H-800), 3.86 (dd, J ¼ 2.4, 11.8 Hz, 1H, H-900b), 3.90 (m, 1H, H-40), 3.96–4.04
(m, 3H, H-2, H-30, H-1000b), 4.09 (dd, J ¼ 3.1, 10.9 Hz, 1H, H-3), 4.18 (m, 1H, H-4),
4.53 (d, J ¼ 7.8 Hz, 1H, H-10), 4.68 (d, J ¼ 8.4 Hz, 1H, H-1); 13C NMR (125 MHz,
D2O): d 2 1.8 (SiMe3), 17.8 (C-2000), 22.2 (CH3), 40.3 (C-300), 52.4 (C-500), 53.9 (C-2),
61.2, 61.4 (C-6, C-60), 62.6 (C-900), 67.5 (C-40), 68.1 (C-4), 68.2 (C-700), 68.7 (C-400),
69.0 (C-1000), 69.4 (C-20), 69.5 (C-600), 72.0 (C-800), 75.2, 75.3 (C-5, C-50), 76.1 (C-30),
78.5 (C-3), 100.5 (2C, C-1, C-100), 104.3 (C-10); HRMS (FAB) Calcd for
C30H51Cl3N2O19Si [M 2 NH4]þ: 894.2264; Found: 894.2261.
2-(Trimethylsilyl)ethyl (5-Acetamido-3,5-dideoxy-D-glycero-a-
D-galacto-2-nonulopyranosylonic acid)-(2!3)-b-D-
Galactopyranosyl-(1!3)-b-D-
galactopyranoside (27)
According to the procedure described for the synthesis of 25, compound 16 (10.0 mg,
22.6 mmol) was incubated with CMP-NeuAc (22.3 mg, 33.9 mmol), BSA (2.3 mg), CIAP
(2 mL) and ST3Gal III (500 mL, 9 U/L). After 17 hr additional ST3Gal III (250 mL) was
added, and the incubation was continued for 3 d. After work-up, the residue was
chromatographed on silica gel (DCM/MeOH/H2O 10 : 4 : 0.4, then 6 : 4 : 1) to yield
starting material 16 (4.0 mg, 40%) and trisaccharide 27 (9.7 mg, 59%) as colorless
powders after a final lyophilization from water.
1
[a]D þ4.1 (c ¼ 0.63, H2O); H NMR (500 MHz, D2O): d 20.02 (s, 9H, SiMe3),
0.97 (m, 1H, H-2000a), 1.07 (m, 1H, H-2000b), 1.79 (t, J ¼ 12.1 Hz, 1H, H-300a), 2.02
(s, 3H, CH3), 2.75 (dd, J ¼ 4.6, 12.4 Hz, 1H, H-300b), 3.58 (dd, J ¼ 1.5, 9.2 Hz, 1H,
H-700), 3.59–3.66 (m, 4H, H-2, H-20, H-600, H-900a), 3.66–3.77 (m, 8H, H-5, H-6, H-50,
H-60, H-400, H-1000a), 3.80 (dd, J ¼ 3,4, 9.9 Hz, 1H, H-3), 3.84–3.88 (m, 3H, H-500, H-800,
H-900b), 3.94 (d, J ¼ 3.0 Hz, 1H, H-40), 4.04 (ddd, J ¼ 5.2, 10.0, 12.7 Hz, 1H, H-1000b),
4.10 (dd, J ¼ 3.1, 9.8 Hz, 1H, H-30), 4.18 (d, J ¼ 3.3 Hz, 1H, H-4), 4.45 (d, J ¼ 8.0 Hz,
1H, H-1), 4.68 (d, J ¼ 7.8 Hz, 1H, H-10); 13C NMR (125 MHz, D2O): d 22.0 (SiMe3),
18.1 (C-2000), 22.6 (CH3), 40.2 (C-300), 52.2 (C-500), 61.4, 61.5 (C-6, C-60), 63.1 (C-900),

ORDER
REPRINTS
22
Schwardt et al.
67.9 (C-40), 68.6 (C-700), 68.8 (C-400), 68.9 (C-1000), 69.0 (C-4), 70.1, 70.3 (C-2, C-20),
72.4 (C-800), 73.4 (C-600), 75.2, 75.4 (C-5, C-50), 76.1 (C-30), 83.1 (C-3), 100.4 (C-10þ0),
102.3 (C-1), 104.6 (C-10); HRMS (FAB) Calcd for C28H51NO19Si [M 2 NH4] :
751.3186; Found: 751.3172.
Attempted Enzymatic Sialylation of 2-(Trimethylsilyl)ethyl b-D-
Galactopyranosyl-(1!3)-[sodium (5-acetamido-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosynate)]-(2!6)-b-D-Galactopyranoside (20)
According to the procedure described for the synthesis of 25, trisaccharide 20
(10.0 mg, 13.2 mmol) was incubated with CMP-NeuAc (13.0 mg, 19.8 mmol), CIAP
(2 mL) and ST3Gal III (500 mL, 9 U/L) in a mixture of 50 mM sodium cacodylate-buffer
pH 6.5 (2 mL), 60 mM aqueous MnCl2 (2 mL) and deionized water (1.3 mL) containing
BSA (2.0 mg). After 24 hr, additional ST3Gal III (250 mL) was added, and the incubation
was continued for 3 d. After work-up, the crude product was chromatographed on silica gel
(DCM/MeOH/H2O 10 : 4 : 0.8) to yield exclusively starting material 20 (9.5 mg, 95%) as
a colorless powder after a final lyophilization from water.
ACKNOWLEDGMENTS
We thank Markus Streiff (Novartis Pharma Inc., Basel) for supplying us with the
ST3Gal III construct. Financial support by the Volkswagen Stiftung is kindly
acknowledged.
REFERENCES
1. Dwek, R.A. Glycobiology: toward understanding the function of sugars. Chem. Rev.
1996, 96, 683–720.
2. McAucliffe, J.C.; Hindsgaul, O. Carbohydrate drugs—an ongoing challenge. Chem.
Ind. 1997, 3, 170–174.
3. Hakomori, S.-I. Structure and function of sphingoglycolipids in transmembrane
signalling and cell–cell interactions. Biochem. Soc. Trans. 1993, 21, 583–595.
4. Varki, A. Biological roles of oligosaccharides: all of the theories are correct.
Glycobiology 1993, 3, 97–130.
5. Hannun, Y.A. Functions of ceramide in coordinating cellular responses to stress.
Science 1996, 274, 1855–1859.
6. Livingston, P.O. Approaches to augmenting the immunogenicity of melanoma
gangliosides: from whole melanoma cells to ganglioside-KLH conjugate vaccines.
Immunol. Rev. 1995, 145, 147–166.
7. (a) Yang, L.J.-S.; Zeller, C.B.; Shaper, N.L.; Kiso, M.; Hasegawa, A.; Shapiro, R.E.;
Schnaar, R.L. Gangliosides are neuronal ligands for myelin-associated glycoprotein.
Proc. Natl. Acad. Sci. USA 1996, 93, 814–818; (b) Vyas, A.A.; Patel, H.V.;
Fromholt, S.E.; Heffer-Lauc, M.; Vyas, K.A.; Dang, J.; Schachner, M.; Schnaar, R.L.
Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein

ORDER
REPRINTS
Recombinant Rat ST3Gal III
23
(MAG), an inhibitor of nerve regeneration. Proc. Natl. Acad. Sci. USA 2002, 99,
8412–8417.
8. (a) Vinson, M.; van der Merwe, P.A.; Kelm, S.; May, A.; Jones, E.Y.; Crocker, P.R.
Characterization of the sialic acid-binding site in sialoadhesin by site-directed
mutagenesis. J. Biol. Chem. 1996, 271, 9267–9272; (b) Crocker, P.R.; Clark, E.A.;
Filbin, M.; Gordon, S.; Jones, Y.; Kehrl, J.H.; Kelm, S.; Le Douarin, N.; Powell, L.;
Roder, J.; Schnaar, R.L.; Sgroi, D.C.; Stamenkovic, K.; Schauer, R.; Schachner, M.;
van den Berg, T.K.; van der Merwe, P.A.; Watt, S.M.; Varki, A. Siglecs: a family of
sialic-acid binding lectins. Glycobiology 1998, 8, v.
9. (a) Tang, S.; Shen, Y.J.; DeBellard, M.B.; Mukhopadhyay, G.; Salzer, J.L.;
Crocker, P.R.; Filbin, M.T. Myelin-associated glycoprotein interacts with neurons via
a sialic acid binding site at ARG118 and a distinct neurite inhibition site. J. Cell Biol.
1997, 138, 1355–1366; (b) Domeniconi, M.; Cao, Z.; Spencer, T.; Sivasankaran, R.;
Wang, K.C.; Nikulina, E.; Kimura, N.; Cai, H.; Deng, K.; Gao, Y.; He, Z.; Filbin, M.T.
Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite
outgrowth. Neuron 2002, 35, 283–290.
10. Khan, S.H.; Hindsgaul, O. Chemical synthesis of oligosaccharides. In Molecular
Glycobiology; Fukuda, M., Hindsgaul, O., Eds.; IRL-Press: Oxford, 1994; 206–229.
11. (a) Gijsen, H.J.M.; Qiao, L.; Fitz, W.; Wong, C.-H. Recent Advances in the
chemoenzymic synthesis of carbohydrates and carbohydrate mimetics. Chem. Rev.
1996, 96, 443–473; (b) Qian, X.; Sujino, K.; Palcic, M.M. Enzymatic glycosylations
with non-natural donors and acceptors. In Carbohydrates in Chemistry and Biology;
Ernst, B., Hart, G.W., Sinay¨, P., Eds.; Wiley-VCH: Weinheim, 2000; Vol. 2,
685–704.
¨
12. (a) Ohrlein, R. Glycosyltransferase-catalyzed synthesis of non-natural oligosacchar-
ides. Topics Curr. Chem. 1999, 200, 227–254; (b) Ernst, B.; Oehrlein, R. Substrate
and donor specificity of glycosyl transferases. Glycoconj. J. 1999, 16, 161–170;
(c) Ohrlein, R. Carbohydrates and derivatives as potential drug candidates with
emphasis on the selectin and linear-b area. Mini Rev. Med. Chem. 2001, 1, 349–361.
13. (a) Tsuji, S.; Datta, A.K.; Paulson, J.C. Systematic nomenclature for sialyltrans-
ferases. Glycobiology 1996, 6, v–xi; (b) Lau, J.T.Y.; Wuensch, S.A. Sialyltrans-
ferases. In Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G.W., Sinay¨, P.,
Eds.; Wiley-VCH: Weinheim, 2000; Vol. 3, 213–226.
14. (a) Harduin-Lepers, A.; Recchi, M.A.; Delannoy, P. 1994, the year of
sialyltransferases. Glycobiology 1995, 5, 741–758; (b) Harduin-Lepers, A.;
Vallejo-Ruiz, V.; Krzewinski-Recchi, M.A.; Samyn-Petit, B.; Julien, S.;
Delannoy, P. The human sialyltransferase family. Biochimie 2001, 83, 727–737.
15. (a) Kitagawa, H.; Paulson, J.C. Differential expression of five sialyltransferase genes
in human tissues. J. Biol. Chem. 1994, 269, 17872–17878; (b) Lee, Y.-C.; Kojima, N.;
Wada, E.; Kurosawa, N.; Nakaoka, T.; Hamamoto, T.; Tsuji, S. Cloning and
expression of cDNA for a new type of Galbl,3GalNAc a2,3-sialyltransferase. J. Biol.
Chem. 1994, 269, 10028–10033.
16. Kim, Y.J.; Kim, K.S.; Kim, S.H.; Kim, C.H.; Ko, J.H.; Choe, I.S.; Tsuji, S.; Lee, Y.C.
Molecular cloning and expression of human Galb,3GalNAc a2,3-sialyltransferase
(hST3Gal II). Biochem. Biophys. Res. Commun. 1996, 228, 324–327.
17. (a) Gambert, U.; Thiem, J. Multi-enzyme system for synthesis of the sialylated
Thomsen-Friedenreich antigen determinant. Eur. J. Org. Chem. 1999, 107–110;

ORDER
REPRINTS
24
Schwardt et al.
´
(b) Dudziak, G.; Bezay, N.; Schwientek, T.; Clausen, H.; Kunz, H.; Liese, A.
Cyclodextrin-assisted glycan chain extension on a protected glycosyl amino acid.
´
Tetrahedron 2000, 56, 5865–5869; (c) Bezay, N.; Dudziak, G.; Liese, A.; Kunz, H.
Chemoenzymatic-chemical synthesis of a (2–3)-sialyl T threonine building block and
its application to the synthesis of the N-terminal sequence of leukemia-associated
leukosialin (CD 43). Angew. Chem. Int. Ed. Engl. 2001, 40, 2292–2295;
(d) Zeng, X.X.; Nakaaki, Y.; Murata, T.; Usui, T. Chemoenzymatic synthesis of
glycopolypeptides carrying a-Neu5Ac-(2!3)-ß-D-Gal-(1!3)-a-D-GalNAc, b-D-
Gal-(1!3)-a-D-GalNAc, and related compounds and analysis of their specific
interactions with lectins. Arch. Biochem. Biophys. 2000, 383, 28–37; (e) Suzuki, K.;
Matsuo, I.; Isomura, M.; Ajisaka, K. Chemoenzymatic synthesis of neuaca-(2!3)-
galb-(1!3)-[neuaca-(2!6)]-galnaca-O-(Z)-serine (N-protected MUC II oligosac-
charide-serine). J. Carbohydr. Chem. 2002, 21, 99–111; (f) Blixt, O.; Allin, K.;
Pereira, L.; Datta, A.; Paulson, J.C. Efficient chemoenzymatic synthesis of O-linked
sialyl oligosaccharides. J. Am. Chem. Soc. 2002, 124, 5739–5746.
18. (a) Wen, D.X.; Livingstone, B.D.; Medzihradszky, K.K.; Kelm, S.; Burlingame, A.L.;
Paulson, J.C. Primary structure of Galß1,3(4)GlcNAc:a2,3-sialyltransferase deter-
mined by mass spectrometry sequence analysis and molecular cloning. J. Biol. Chem.
1992, 267, 21011–21019; (b) Kitagawa, H.; Paulson, J.C. Cloning and expression of
human Galß1,3(4)GlcNAc:a2,3-sialyltransferase. Biochem. Biophys. Res. Commun.
1993, 194, 375–382; (c) Williams, M.A.; Kitagawa, H.; Datta, A.K.; Paulson, J.C.;
Jamieson, J. Large-scale expression of recombinant sialyltransferases and comparison
of their kinetic properties with native enzymes. Glycoconjugate J. 1995, 12, 755–761.
19. (a) Palcic, M.M.; Venot, A.P.; Ratcliffe, R.M.; Hindsgaul, O. Enzymic synthesis of
oligosaccharides terminating in the tumor-associated sialyl-Lewisa determinant.
Carbohydr. Res. 1989, 190, 1–11; (b) Liu, Y.C.; Li, H.; Otter, A.; Kamath, V.P.;
Streiff, M.B.; Palcic, M.M. Chemo-enzymatic synthesis of trimeric sialyl Lewisx
¨
pentadecasaccharide. Can. J. Chem. 2002, 80, 540–545; (c) Baisch, G.; Ohrlein, R.
Chemoenzymatic synthesis of sialyl Lewisx glycopeptides. Angew. Chem. Int. Ed.
Engl. 1996, 35, 1812–1815; (d) Zeng, S.; Gallego, R.G.; Dinter, A.; Mallisard, M.;
Kamerling, J.P.; Vliegenthart, J.F.G.; Berger, E.G. Complete enzymic synthesis of the
mucin-type sialyl Lewisx epitope, involved in the interaction between PSGL-1 and
P-Selectin. Glycoconj. J. 2000, 16, 487–497.
20. (a) Ichikawa, Y.; Lin, Y.-C.; Dumas, D.P.; Shen, G.-J.; Garcia-Junceda, E.;
Williams, M.A.; Bayer, R.; Ketcham, C.; Walker, L.E.; Paulson, J.C.; Wong, C.-H.
Chemical-enzymatic synthesis and conformational analysis of sialyl Lewisx and
derivatives. J. Am. Chem. Soc. 1992, 114, 9283–9298; (b) Koeller, K.M.; Wong, C.-H.
Chemoenzymatic synthesis of sialyl-trimeric-Lewisx. Chem. Eur. J. 2000, 6,
1243–1251.
21. Duclos, R.I. The total synthesis of ganglioside GM3. Carbohydr. Res. 2000, 328,
489–507; and references cited.
22. Kitagawa, H.; Paulson, J.C. Cloning of a novel a2,3-sialyltransferase that sialylates
glycoprotein and glycolipid carbohydrate groups. J. Biol. Chem. 1994, 269, 1394–1401.
23. Ishii, A.; Ohta, M.; Watanabe, Y.; Matsuda, K.; Ishiyama, K.; Sakoe, K.;
Nakamura, M.; Inokuchi, J.; Sanai, Y.; Saito, M. Expression, cloning and functional
characterization of human cDNA for ganglioside GM3 synthase. J. Biol. Chem. 1998,
273, 31652–31655.

ORDER
REPRINTS
Recombinant Rat ST3Gal III
25
24. (a) Melkerson-Watson, L.C.; Sweeley, C.C. Purification to apparent homogeneity by
immunoaffinity chromatography and partial characterization of the GM3 ganglioside-
forming enzyme, CMP-sialic acid:lactosylceramide a2,3-sialyltransferase (SAT-1),
from rat liver golgi. J. Biol. Chem. 1991, 266, 4448–4457; (b) Preuss, U.; Gu, X.;
Gu, T.; Yu, R.K. Purification and characterization of CMP-N-acetylneuraminic
acid:lactosylceramide (a2–3)sialyltransferase (GM3-synthase) from rat brain. J. Biol.
Chem. 1993, 268, 26273–26278.
25. Okajima, T.; Fukumoto, S.; Miyazaki, H.; Ishida, H.; Kiso, M.; Furukawa, K.;
Urano, T.; Furukawa, K. Molecular cloning of a novel a2,3-sialyltransferase (ST3Gal
VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids.
J. Biol. Chem. 1999, 274, 11479–11486.
26. Palcic, M.M.; Hindsgaul, O. Glycosyltransferases in the synthesis of oligosaccharide
analogs. Trends Glycosci. Glycotechnol. 1996, 8, 37–49.
27. Ito, Y.; Gaudino, J.J.; Paulson, J.C. Synthesis of bioactive sialosides. Pure Appl.
Chem. 1993, 65, 753–762.
28. van Dorst, J.A.L.M.; Tikkanen, J.M.; Krezdorn, C.H.; Streiff, M.B.; Berger, E.G.; van
Kuik, J.A.; Kamerling, J.P.; Vliegenthart, J.F.G. Exploring the substrate specificities
of a-2,6 and a-2,3-sialyltransferases using synthetic acceptor analogues. Eur. J.
Biochem. 1996, 242, 674–681.
¨
29. (a) Baisch, G.; Ohrlein, R.; Streiff, M.; Ernst, B. Enzymatic a(2–3)-sialylation of
non-natural disaccharides with cloned sialyl-transferase. Bioorg. Med. Chem. Lett.
¨
1996, 6, 755–758; (b) Baisch, G.; Ohrlein, R.; Streiff, M. Enzymatic a(2–3)-
sialylation of non-natural type-I (Lewisc) disaccharides with recombinant sialyl-
¨
transferase. Bioorg. Med. Chem. Lett. 1998, 8, 157–160; (c) Baisch, G.; Ohrlein, R.
Glycosyl-transferase catalyzed assemblage of sialyl-Lewisx-saccharopeptides.
¨
Bioorg. Med. Chem. 1998, 6, 1673–1682; (d) Baisch, G.; Ohrlein, R. Glycosyl-
transferase catalyzed assemblage of sialyl-Lewisa saccharopeptides. Carbohydr. Res.
1998, 312, 61–72.
30. Burger, P.C.; Lotscher, M.C.; Streiff, M.C.; Kleene, R.; Kaissling, B.; Berger, E.G.
Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in
cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization.
Glycobiology 1998, 8, 245–257.
31. Gosselin, S.; Alhussaini, M.; Streiff, M.B.; Takabayashi, K.; Palcic, M.M.
A continuous spectrophotometric assay for glycosyltransferases. Anal. Biochem.
1994, 220, 92–97.
32. (a) Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. The use of 2-deoxy-2-trichloroacetamido-
d-glucopyranose derivatives in syntheses of oligosaccharides. Carbohydr. Res. 1994,
260, 189–202; (b) Belot, F.; Jacquinet, J.-C. Intermolecular aglycon transfer of a
phenyl 1-thiogalactosaminide derivative under trichloroacetimidate glycosylation
conditions. Carbohydr. Res. 1996, 290, 79–86.
33. Biessen, E.A.L.; Beuting, D.M.; Roelen, H.C.P.F.; van de Marel, G.A.; van
Boom, J.H.; van Berkel, T.J.C. Synthesis of cluster galactosidases with high affinity
for the hepatic asialoglycoprotein receptor. J. Med. Chem. 1995, 38, 1538–1546.
34. Jansson, K.; Ahifors, S.; Frejd, T.; Kihlberg, J.; Magnusson, G.; Dahmen, J.; Noori, G.;
Stenvall, K. 2-(Trimethylsilyl)ethyl glycosides. 3. synthesis, anomeric deblocking,
and transformation into 1,2-trans 1-O-acyl sugars. J. Org. Chem. 1988, 53,
5629–5647.

ORDER
REPRINTS
26
Schwardt et al.
35. Murase, T.; Kameyama, A.; Kartha, K.P.R.; Ishida, H.; Kiso, M.; Hasegawa, A.
Synthetic studies on sialoglycoconjugates 5: a facile, regio and stereoselective
synthesis of ganglioside GM4 and its position isomer. J. Carbohydr. Chem. 1989, 8,
265–283.
36. Fu¨tigedi, P.; Garegg, P.J. A novel promoter for the efficient construction of 1,2-trans-
linkages in glycoside synthesis, using thioglycosides as glycosyl donors. Carbohydr.
Res. 1986, 149, c9–c12.
37. Kanie, O.; Kiso, M.; Hasegawa, A. Glycosylation using methylthioglycosides of
N-acetylneuraminic acid and dimethyl(methylthio)sulfonium triflate. J. Carbohydr.
Chem. 1986, 7, 501–506.
38. Palcic, M.M. Glycosyltransferases in glycobiology. Meth. Enzymol. 1994, 230,
300–316.
¨
39. Ernst, B.; Wagner, B.; Baisch, G.; Katopodis, A.; Winkler, T.; Ohrlein, R. Substrate
specificity of fucosyl transferase III: an efficient synthesis of sialyl Lewisx-, sialyl
Lewisa derivatives and mimetics thereof. Can. J. Chem. 2000, 78, 892–904.
¨
40. Baisch, G.; Ohrlein, R.; Ernst, B. Enzymatic galactosylation of non-natural
glucosamide-acceptors. Bioorg. Med. Chem. Lett. 1996, 6, 749–754.
¨
41. Baisch, G.; Ohrlein, R.; Streiff, M.; Kolbinger, F. On the preparative use of
recombinant b(1–3)galactosyl-transferase. Bioorg. Med. Chem. Lett. 1998, 8,
751–754.
´
42. Lubineau, A.; Auge, C.; Franc¸ois, P. The use of porcine liver (2!3)-
a-sialyltransferase in the large-scale synthesis of a-Neup5Ac-(2!3)-b-D-Galp-
(1!3)-D-GlcpNAc, the epitope of the tumor-associated carbohydrate antigen CA 50.
Carbohydr. Res. 1992, 228, 137–144.
Received December 20, 2002
Accepted September 15, 2003

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