Simplifying oligosaccharide synthesis: Efficient synthesis of lactosamine and siaylated lactosamine oligosaccharide donors

Article abstract of DOI:10.1021/jo026569v

A practical sequence is described for converting D-glucosamine into peracetylated Gal(β-1,4)-GlcNTroc(β1-S)Ph and Neu5Ac(α-2,3)Gal(β-1,4)GlcNTroc(β1-S)Ph building blocks using a synthetic strategy based on chemoenzymatic oligosaccharide synthesis. The known trichloroethoxycarbonyl, N-Troc, protecting group was selected as a suitable protecting group for both enzymatic and chemical reaction conditions. These oligosaccharide building blocks proved effective donors for the β-selective glycosylation of the unreactive OH-3 of a polymeric PEG-bound acceptor and for the axial OH-2 of a mannose acceptor in good yields. The resulting complex oligosaccharides are useful for vaccine and pharmaceutical applications.

Full text of DOI:10.1021/jo026569v

Sim p lifyin g Oligosa cch a r id e Syn th esis: Efficien t Syn th esis of
La ctosa m in e a n d Sia yla ted La ctosa m in e Oligosa cch a r id e Don or s
Fengyang Yan, Seema Mehta, Eva Eichler, Warren W. Wakarchuk, Michel Gilbert,
Melissa J . Schur, and Dennis M. Whitfield*
Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Room 3024,
Ottawa, Ontario, K1A 0R6, Canada
dennis.whitfield@nrc.gc.ca
Received October 15, 2002
A practical sequence is described for converting D-glucosamine into peracetylated Gal(â-1,4)-
GlcNTroc(â1-S)Ph and Neu5Ac(R-2,3)Gal(â-1,4)GlcNTroc(â1-S)Ph building blocks using a synthetic
strategy based on chemoenzymatic oligosaccharide synthesis. The known trichloroethoxycarbonyl,
N-Troc, protecting group was selected as a suitable protecting group for both enzymatic and chemical
reaction conditions. These oligosaccharide building blocks proved effective donors for the â-selective
glycosylation of the unreactive OH-3 of a polymeric PEG-bound acceptor and for the axial OH-2 of
a mannose acceptor in good yields. The resulting complex oligosaccharides are useful for vaccine
and pharmaceutical applications.
Oligosaccharides play an important role in biological
systems, such as cell-cell interaction, cell adhesion, and
immunogenic recognition.1 To further understand and
exploit the activities of oligosaccharides, access is re-
quired to natural and nonnatural oligosaccharides, yet
the synthesis of reasonable quantities of complex oli-
gosaccharides remains one of the most challenging areas
of chemistry.2 One of the most efficient ways to synthesize
oligosaccharides is to prepare di-, tri-, and higher oli-
gosaccharide building blocks and then convergently as-
semble the large oligosaccharide from these building
blocks.3 Often, these building blocks contain oligosaccha-
rides with cis linkages or other “difficult” linkages such
as those with 2-keto-acids such as sialic acids that
frequently can only be formed in low yield, and the
reactions often lead to complex mixtures that require
careful separations.4 To assemble these building blocks,
high-yielding glycosylation reactions that form trans
linkages via neighboring group participation are typically
used.5
available methods for the syntheses of lactosaminyl
donors such as 1 are few in number, and all syntheses
require many chemical steps with low overall yields.7 An
alternative to the chemical procedures for oligosaccharide
synthesis is given by protocols based on the use of
glycosyltransferases for carbohydrate assemblage since
glycosyltransferases are highly stereo- and regioselective
with regard to the glycoside bond formations, and no
tedious protection/deprotection steps are required.8 On
the other hand, with the recent emergence of a wide
variety of cloned and expressed glycosyltransferases from
bacterial sources, many enzymes are now available in
synthetically useful quantities.9 Similarly, the requisite
nucleotide donors are also becoming available in bulk at
competitive prices.10 Following our earlier examples for
the synthesis of oligosaccharides donors,8b,c we describe
here a practical chemoenzymatic methodology to per-
acetylated Gal(â-1,4)GlcNTroc(â1-S)Ph 1b and Neu5Ac-
(R-2,3)Gal(â-1,4)GlcNTroc(â1-S)Ph 2b building block do-
nors from 2-amino-2-deoxy-D-glucosamine 3. Their subse-
quent use for the efficient generation of complex oligosac-
charides is also reported.
N-Acetylated lactosamine (Gal(â-1,4)GlcNAc) and its
sialylated (usually R2,3 or R2,6) extension are ubiquitous
components of cell surface glycans.6 Thus, an efficient
method to large-scale amounts of these compounds, as
shown in Scheme 1, in a form suitable for further
glycosylation chemistry is highly desirable. However, the
The key to this development is the choice of N-
protecting group on the glucosamine since it must be
capable of neighboring group participation to ensure high
â-selectivity in the glycosylation reaction and stable to
both enzymatic and chemical reaction conditions. N-
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2000, 2, 751.
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(10) Bulter, T.; Elling, L. Glycoconjugate J . 1999, 16, 147.
10.1021/jo026569v CCC: $25.00 Published 2003 American Chemical Society
Published on Web 02/21/2003
2426
J . Org. Chem. 2003, 68, 2426-2431

Simplifying Oligosaccharide Synthesis
SCHEME 1
SCHEME 2
Phthalimide is an obvious choice since it is widely used
in chemical glycosylation reactions.11 We started a pro-
gram to prepare N-phthaloyl (N-Phth) lactosamine 1a
and (R-2,3) sialylated lactosamine 2a building blocks from
GlcNPhthâ1-SPh 5a (Scheme 2).12 Eventually, it was
found that the GlcNPhthâ1-SPh 5a solubilized with a
small amount of DMF (∼1% V/V) was a substrate for the
available galactosyltransferase and sialyltransferase (see
the Experimental Section) and after acetylation was
turned into potential donor 8a ; see Schemes 3 and 4. But,
the overall yields were always low, even though TLC
monitoring of the enzyme reactions suggested that all the
substrates were consumed. The products showed (M +
18)+ peaks in their MS spectra. Several stability studies
at different pHs were monitored by 1H NMR and typically
showed the disappearance of the anomeric proton peak
(δ 5.69 ppm) of the substrate 5a and the appearance of
a new anomeric peak (δ 5.06 ppm, see the Supporting
Information). From these data, it was concluded that the
N-Phth ring opened in water and that the opening is
accelerated at high pH. The use of p-methoxybenzyl or
even better p-nitrobenzyl glycosides of the N-Phth glu-
cosamine minimized but did not eliminate this side
reaction (not shown). With the small quantities of 8a
available, several glycosylations with polymer-bound
acceptors such as compound 10, that worked well with
phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-â-
D-glucopyranoside as donor,13 failed or gave only low
yields. Therefore, we decided to investigate other N-
protecting groups.
2,5-Dimethylpyrrole (N-DMP) group14 is a stable and
soluble N-protecting group in both chemical and enzy-
matic reaction conditions. However, the peracetylated
N-DMP-protected thiophenyl and trichloroacetimidate
glycoside as donors were not reactive with low nucleo-
philicity acceptors such as the unreactive HO-3 position
of compound 10; see Scheme 5.
Further surveys of N-protecting groups led us to
trichloroethoxycarbonyl (Troc) as a suitable protecting
group, since the N-Troc group has proved to be useful
with respect to facile introduction, deprotection, and
â-selective glycosidation as well as increased reactivity
of donors with a C-2 N-Troc group compared with N-Phth
group.15 In addition, the N-Troc group is stable under a
range of standard conditions used for chemical and
enzymatic oligosaccharide synthesis, although it is sensi-
tive to alcoholysis under basic conditions. The known
thiophenyl glycoside of GlcNTroc 5b as substrate was
readily prepared in a good yield in four steps from
D-glucosamine 3 as shown in Scheme 2.16 Treatment with
inexpensive UDP-glucose and a GalE-Glc/Gal epimerase
fusion enzyme17 led to 89% conversion to the desired
lactosamine derivative Gal(â-1,4)GlcNTroc(â1-S)Ph 6b.
This disaccharide 6b was easily acetylated with acetic
(14) Bowers, S. G.; Coe, D. M.; Boons, G.-J . J . Org. Chem. 1998, 63,
4570.
(11) Debenham, J .; Rodebaugh, R.; Fraser-Reid, B. Liebigs Ann./
Recueil 1997, 791.
(12) Robina, I.; Gomez-Bujedo, S.; Fernandez-Bolanos, J . G.; Pozp,
L. D.; Demange, R.; Picasso, S.; Vogel, P. Carbohydr. Lett. 2000, 3,
389.
(15) A) Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.; Schmidt,
R. R. Carbohydr. Res. 1996, 296, 135. (b) Ellervik, U.; Magnusson, G.
Carbohydr. Res. 1996, 280, 251.
(16) Nilsson, K. G. I. Tetrahedron Lett. 1997, 38, 133.
(17) Yan, F.; Wakarchuk, W. W.; Gilbert, M.; Richards, J . C.;
Whitfield, D. M. Carbohydr. Res. 2000, 328, 3.
(13) Mehta, S.; Whitfield, D. M. Tetrahedron 2000, 56, 6415.
J . Org. Chem, Vol. 68, No. 6, 2003 2427

Yan et al.
SCHEME 3
SCHEME 4
anhydride in the presence of pyridine to give a versatile
lactosaminyl donor 1b in high yield. The 1H and 13C NMR
data clearly indicated the new Gal(â-1,4)-linkage notably
Gal H-4 δ 1H ) 5.34 ppm J 3,4 ) 2.7 Hz and Gal H-1 δ 1H
) 4.48 ppm J 1,2 ) 7.8 Hz. Further sialylation of the
disaccharide 6b with a solution of CMP-Neu5Ac, which
was generated with sialic acid and CTP from a CMP-
synthetase at high pH (8.5),18 and a sialyltransferase
(FUS-01/2)19 at low pH (6.0) generated Neu5Ac(R-2,3)-
Gal(â-1,4)GlcNTroc(â1-S)Ph 7b in nearly quantitative
yield. Unlike our previous report17 in which the expensive
nucleotide sugar donor CMP-Neu5Ac was generated in
situ by a fusion enzyme, this time it was prepared using
a CMP-Neu5Ac synthetase along with sialic acid and
CTP. The supernatant after centrifugation was used
without further purification. This procedure is more
efficient since both the CMP-synthetase and sialyltrans-
ferase are used at their pH optimum (see above). This
(18) Karwaski, M.-F.; Wakarchuk, W. W.; Gilbert, M. Protein
Expression Purif. 2002, 25, 237.
2428 J . Org. Chem., Vol. 68, No. 6, 2003

Simplifying Oligosaccharide Synthesis
SCHEME 5
SCHEME 6
means that less enzyme and less nucleotide sugar donor
are needed for the same amount of product.
For the synthesis of final sialylated lactosamine donor
2b we treated compound 7b with acetic anhydride/
pyridine at 45 °C to give a desired intra 1,2-lactone 8b
which was indicated by 1H, 13C, and HMBC NMR spectra,
notably the long range connectivity between the lactone
CdO at δ 13C ) 163.12 ppm and Gal H-2 at δ 1H ) 4.68
ppm. Treatment of 1,2-lactone 8b with DMAP in dry
methanol and then reacetylation with acetic anhydride/
pyridine8c gave a complex mixture from which 2b was
isolated as the main product in less than 10% yield. The
sensitivity of the N-Troc group to methanol under basic
condition may be the cause of this problem. A shorter
alternative route was developed that involves esterifica-
tion of compound 7b with an ion-exchange resin (H+
form) in methanol followed by acetylation to give a
reasonable yield (74%) of trisaccharide donor 2b ac-
companied by small amounts of lactone 9, as shown in
Scheme 4. Lactone 9 is chromatographically and spec-
troscopically different from 8b but its definitive struc-
tural determination is in progress. It should be noted that
the processing of compound 2b was greatly simplified by
using acetic anhydride as the solvent and Sc(OTf)3 as the
acetylation catalyst. This process is very simple and
readily scalable as the product is isolated by aqueous
organic solvent extractions followed by flash silica gel
chromatography.
1A Group B Streptococcus capsular polysaccharide of
interest in our institute for vaccine development.23 Simi-
larly, the trisaccharide donor 2b was reacted with the
axial OH-2 of mannose acceptor 13 to yield tetrasaccha-
ride 14 in 75% yield; see Scheme 6. The 1D and 2D NMR
spectroscopy (gCOSY, HSQC, HMBC) of 14 indicated that
the glycosidic linkage is a trans GlcNTroc(â-1,2)-Manp
linkage (chemical shift of the GlcN-Troc anomeric proton
is δ 1H ) 4.81 ppm, J > 7.0 Hz). Standard one-step
deprotection and N-acetylation of the N-Troc with Zn in
acetic anhydride gave 15.15a Subsequently the O-benzyl
groups were removed by hydrogenation over Pd/C and
the acyl groups by transesterification followed by hy-
drolysis to yield 18 as shown in Scheme 6. The three
anomerics at GlcN δ 1H ) 4.60 ppm J 1,2 ) 8.0 Hz, Gal δ
To demonstrate the efficiency of these compounds
lactosaminyl donor 1b was successfully reacted with the
unreactive OH-3 of acceptor 10 which is bound to
the polymer poly(ethylene glycol) (MPEG), CH3O(CH2-
CH2O)n-H via the linker dioxyxylene [DOX, -(O)CH2-
PhCH2(O)-]20 to yield the â-linked trisaccharide 11. The
small electron donating 1-methyl 1′-cyclopropylmethyl,
MCPM, protecting group was used to activate the O-3.21
The successful glycosylation with 1b but not with 1a
perhaps reflects the 30-fold greater reactivity of N-Troc
versus N-Phth reported for monosaccharide donors.22
Donors 2a , 2b, and 2c are all 3,4,6-triacetyl protected
and therefore deactivated and thus require strong pro-
moters. After Sc(OTf)3-mediated cleavage, the trisac-
charide 12 was isolated without affecting the N-Troc
group as evidenced by the CdO δ 13C ) 153.71 ppm and
1
1H ) 4.56 ppm J 1,2 ) 8.0 Hz, and Man δ H ) 4.80 ppm
J 1,2 ) 1.0 Hz support the expected anomericities. Tet-
rasaccharide 18 is a model of a typical arm of an N-linked
glycopeptide.24
In conclusion, the large-scale synthesis of peracetylated
Gal(â-1,4)GlcNTroc(â1-S)Ph 1b and Neu5Ac(R-2,3)Gal-
(â-1,4)GlcNTroc(â1-S)Ph 2b as effective building blocks
was accomplished through enzymatic and chemical trans-
formation of D-glucosamine by a short route with good
yields. The key to this development was the choice of the
N-Troc group since it is capable of neighboring group
participation to ensure high â-selectivity in subsequent
glycosylation reactions and stable to both enzymatic and
chemical reaction conditions. Both building blocks have
1
the CdOOCH2 δ H ) 4.52 and 4.28 ppm, J ) 12.7 Hz;
see Scheme 5. This trisaccharide â-12 is part of the type
(19) Gilbert, M.; Bayer, B.; Cunningham, A.-M.; DeFrees, S.; Gao,
Y.; Watsor, D. C.; Young, N. M.; Wakarchuk, W. W. Nature Biotech-
nol.1998, 16, 769.
(20) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J . J . J . Am. Chem.
Soc. 1995, 117, 2116.
(21) Eichler, E.; Yan, F.; Sealy, J .; Whitfield, D. M. Tetrahdron 2001,
57, 6679.
(23) J ennings, H. J . Curr. Top. Microbiol. 1990, 150, 97.
(24) (a) Sherman, A. A.; Yudina, O. N.; Shashkov, A. S.; Menshov,
V. M.; Nifantev, N. E. Carbohydr. Res. 2002, 337, 451. (b) Seifert, J .;
Ogawa, T.; Kurono, S.; Ito, Y. Glycoconjugate J . 2000, 17, 407.
(22) Koeller, K. M.; Wong, C.-H. Chem. Rev. 2000, 100, 4465.
J . Org. Chem, Vol. 68, No. 6, 2003 2429

Yan et al.
H, J ) 12.5 Hz, 1/2 CH2CCl3), 4.95 (d, 1 H, J ) 10.5 Hz,
H-1GlcN), 4.75 (d, 1 H, J ) 12.0 Hz, 1/2 CH2CCl3), 4.57 (d, 1 H,
J ) 8.0 Hz, H-1Gal), 4.13 (dd, 1 H, J ) 10.0, 2.5 Hz, H-3Gal),
4.00 (dd, 1 H, J ) 12.0 Hz, J < 1.0 Hz, H-6GlcN), 3.97 (d, 1 H,
J ) 2.5 Hz, H-4Gal), 3.88-3.92 (m, 2 H, H-8Neu, H-9Neu), 3.87
(dd, 1 H, J ) 12.5 Hz, J ) 4.5 Hz, H-6GlcN), 3.86 (t, 1 H, J )
10.0 Hz, H-5Neu), 3.78 (t, 1 H, J ) 8.5 Hz, H-4GlcN), 3.76 (t, 1
H, J ) 9.5 Hz, H-3GlcN), 3.71-3.75 (m, 3 H, 2 × H-6Gal, H-5Gal),
3.70 (td, 1 H, J ) 11.5, 4.5 Hz, H-4Neu), 3.67 (m, 1 H, H-9Neu),
3.65 (dd, 1 H, J ) 10.5, 1.5 Hz, H-6Neu), 3.62-3.64 (brs, 1 H,
H-5GlcN), 3.61 (t, 1 H, J ) 8.0 Hz, H-2GlcN), 3.60 (d, 1 H, J )
8.0 Hz, H-7Neu), 3.58 (dd, 1 H, J ) 10.0, 8.0 Hz, H-2Gal), 2.77
(dd, 1 H, J ) 12.5, 5.0 Hz, H-3eqNeu), 2.04 (s, 3 H, OdCCH3),
1.81 (t, 1 H, J ) 12.5 Hz, H-3axNeu); 13C NMR (D2O) δ 175.71,
174.57 (C-1Neu, OdCCH3), 157.16 (OdCOCH2CCl3), 132.96,
132.21, 130.04, 128.83 (6 × C, Ph), 103.24 (C-1Gal), 100.50
(C-2Neu), 95.63 (CCl3), 87.59 (C-1GlcN), 79.57 (C-5GlcN), 78.63
(C-4GlcN), 76.17 (C-3Gal), 75.88 (C-5Gal), 74.95 (CH2CCl3), 74.48
(C-3GlcN), 73.57 (C-6Neu), 72.46 (C-8Neu), 70.07 (C-2Gal), 69.03
(C-4Neu), 68.78 (C-7Neu), 68.18 (C-4Gal), 63.27 (C-9Neu), 61.74
(C-6Gal), 60.80 (C-6GlcN), 56.78 (C-2GlcN), 52.37 (C-5Neu), 40.32
been used for the synthesis of oligosaccharides for vaccine
and pharmaceutical applications. In principle, the syn-
thetic strategy disclosed in this paper will be applicable
to the synthesis of a variety of di-, tri-, and oligosaccha-
ride donors. Further applications of this methodology are
in progress and will be reported in due course.
Exp er im en ta l Section
Optical rotations were obtained (λ ) 589 nm) at 20 °C in a
10 cm 1 mL cell. NMR spectra were recorded on a 500 or 200
MHz instrument at 300 K. Chemical shifts were given in ppm
relative to the signal of internal TMS or indirectly to solvent
signals 7.26 (CDCl3) or 4.79 (D2O) for 1H NMR spectra and to
the solvent signals 77.0 (CDCl3) or 49.15 (internal methyl
alcohol) for 13C NMR spectra. All signal assignments were
made by standard 1H-1H-COSY and 1H-decoupled 13C-1H-
COSY experiments. All chemicals for synthesis were purchased
from commercial suppliers, and solvents were purified accord-
ing to standard procedures. Silica gel (230-400 mesh) was
used for flash chromatography. C18 silica gel (10% capped with
TMS, 35-70 mesh) was used for reversed-phase chromatog-
raphy.
(C-3Neu), 22.72 (OdCCH3); MS (FAB) calcd for C32H45Cl3N2O19
921.1, found m/z 921.1 [M+].
S
P h en yl O-(Meth yl 5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-
3,5-d id eoxy-D-glycer o-r-D-ga la ct o-2-n on u lop yr a n osylo-
n a te)- (2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-
(1f4)-2-d eoxy-3,6-d i-O-a cet yl-1-t h io-2-(2,2,2-t r ich lor o-
eth oxyca r bon yla m in o)-â-D-glu cop yr a n osid e (2b). A solu-
tion of 7b (280 mg, 0.34 mmol) in 20 mL of dry methanol was
stirred under argon with pretreated DOWEX500Wx8-200 resin
(H+ form) (4 g) at room temperature for 16 h. The resin was
removed by filtration. The filtrate was concentrated, and the
residue was coevaporated with toluene (3 × 20 mL) and dried
under high vacuum to give the methyl ester of 7b. To a solution
of the crude methyl ester in acetic anhydride (10 mL) was
added scandium triflate (15 mg, 0.03 mmol), and the reaction
mixture was stirred at rt for 2 h under argon. The reaction
was quenched with NaHCO3 (10 mg, 0.12 mmol), and product
was extracted with CH2Cl2 (100 mL) three times. The com-
bined organic layer was washed with aqueous NaHCO3, brine,
and water successively, dried over Na2SO4, and evaporated.
The residue was purified by silica gel chromatography (ethyl
acetate-methanol 98:2) to yield pure 2b (305 mg, 74%) and
En zym a tic Syn th esis of P h en yl O-(â-D-Ga la ctop yr a n o-
syl)-(1f4)-2-d e oxy-1-t h io-2-(2,2,2-t r ich lor oe t h oxyca r -
bon yla m in o)-â-D-glu cop yr a n osid e (6b). A fusion enzyme
UDP-galactose 4-epimerase/â(1f 4)galactosyltransferase (GalE-
LgtB, 26.4 units, 24 mL) was added to a solution of 3.2 mM
acceptor 5b (1.0 g), 15 mM MnCl2 (105 mL of 100 mM), and
1.28 mM UDP-Glc (6.0 mL of 150 mM) in 50 mM HEPES
buffer (35 mL of 1 M, pH 7.4). The reaction was performed at
37 °C for a total of 20 h while two additional portions of 1.28
mM UDP-Glc (6.0 mL of 150 mM) were added in 30 min
intervals. The formation of 6b (Rf 0.43) was monitored by TLC
(MeOH-CHCl3-0.5% CaCl2 40:50:10). Chromatography of the
crude reaction mixture on
a C18 reversed-phase column
(water-MeOH 1:1) afforded pure 6b (1.22 g, 89%): [R]D -5.6
1
(c 0.4, CH3OH); H NMR (methanol-d4) δ 7.52-7.55 (m, 2 H,
Ph), 7.26-7.35 (m, 3 H, Ph), 4.91 (d, 1 H, J ) 12.0 Hz, 1/2
CH2CCl3), 4.82 (d, 1 H, J ) 10.5 Hz, H-1GlcN), 4.76 (d, 1 H, J
) 12.0 Hz, 1/2 CH2CCl3), 4.42 (d, 1 H, J ) 7.5 Hz, H-1Gal),
3.95 (dd, 1 H, J ) 12.5, 2.5 Hz, H-6GlcN), 3.88 (dd, 1 H, J )
12.5, 4.0 Hz, H-6GlcN), 3.83 (d, 1 H, J ) 3.0 Hz, H-4Gal), 3.80
(dd, 1 H, J ) 11.5, 7.5 Hz, H-6Gal), 3.70 (dd, 1 H, J ) 11.5, 4.0
Hz, H-6Gal), 3.66-3.69 (m, 2 H, H-4GlcN, H-3GlcN), 3.59-3.62 (m,
1 H, H-5Gal), 3.57 (t, 1 H, J ) 9.5 Hz, H-2GlcN), 3.55 (brt, 1 H,
J ) 8.5 Hz, H-2Gal), 3.50 (dd, 1 H, J ) 9.5, 3.0 Hz, H-3Gal),
3.46 (brs, 1 H, H-5GlcN); 13C NMR (methanol-d4) δ 157.49
(OdCOCH2CCl3), 136.33, 133.43, 130.72, 129.28 (6 × C, Ph),
105.79 (C-1Gal), 98.02 (CCl3), 89.56 (C-1GlcN), 81.49 (C-5GlcN),
81.18 (C-4GlcN), 78.03 (C-5Gal), 76.42 (2 × C, CH2CCl3, C-3GlcN),
75.66 (C-3Gal), 73.48 (C-2Gal), 71.22 (C-4Gal), 63.47 (C-6Gal), 62.81
1
lactone 9 (10 mg, 2%). 2b: [R]D -5.1 (c 0.5, CHCl3); H NMR
(CDCl3) δ 7.49 (m, 2 H, Ph), 7.29 (m, 3 H, Ph), 5.54 (ddd, 1 H,
J ) 9.2, 5.6, 2.8 Hz, H-8Neu), 5.39 (dd, 1 H, J ) 9.2 , 2.8 Hz,
H-7Neu), 5.25 (d, 1 H, J ) 9.6 Hz, NHGlcN), 5.11 (t, 1 H, J ) 9.6
Hz, H-3GlcN), 5.08 (d, 1 H, J ) 10.0 Hz, NHNeu), 4.93 (dd, 1 H,
J ) 10.4, 8.0 Hz, H-2Gal), 4.88 (td, 1 H, J ) 11.2, 4.8 Hz, H-4Neu),
4.87 (d, 1 H, J ) 3.6 Hz, H-4Gal), 4.80 (d, 1 H, J ) 12.4 Hz, 1/2
CH2CCl3), 4.72 (d, 1 H, J ) 12.4 Hz, 1/2 CH2CCl3), 4.69 (d, 1
H, J ) 8.0 Hz, H-1Gal), 4.68 (d, 1 H, J ) 10.4 Hz, H-1GlcN), 4.52
(dd, 1 H, J ) 10.0, 3.2 Hz, H-3Gal), 4.50 (dd, 1 H, J ) 11.6, 2.0
Hz, H-6GlcN), 4.42 (dd, 1 H, J ) 12.4, 2.8 Hz, H-9Neu), 4.19 (dd,
1 H, J ) 12.0, 6.4 Hz, H-6GlcN), 4.04 (t, 1 H, J ) 10.0 Hz,
H-5Neu), 4.00 (m, 2 H, 2 × H-6Gal), 3.98 (dd, 1 H, J ) 12.4, 5.6
Hz, H-9Neu), 3.85 (m, 1 H, H-5Gal), 3.84 (s, 3 H, COOCH3), 3.81
(m, 1 H, H-4GlcN), 3.78 (t, 1 H, J ) 10.0 Hz, H-2GlcN), 3.64 (m,
1 H, H-5GlcN), 3.63 (dd, 1 H, J ) 10.8, 2.8 Hz, H-6Neu), 2.58
(dd, 1 H, J ) 12.8, 4.8 Hz, H-3eqNeu), 2.24, 2.16, 2.09, 2.09, 2.08,
2.05, 2.05, 2.04, 2.00, 1.85 (s, 30 H, OdCCH3), 1.81 (t, 1 H, J
) 12.5 Hz, H-3axNeu); 13C NMR (CDCl3) δ 170.88, 170.65,
170.56, 170.41, 170.39, 170.32, 170.31, 170.26, 170.14, 169.59
(10 × C, OdCCH3), 167.91 (C-1Neu), 154.20 (OdCOCH2CCl3),
132.38, 131.71, 128.86, 127.96 (6 × C, Ph), 101.01 (C-1Gal),
96.74 (C-2Neu), 95.28 (CCl3), 87.10 (C-1GlcN), 77.25 (C-5GlcN),
76.25 (C-4GlcN), 74.52 (CH2CCl3), 73.97 (C-3GlcN), 72.01 (C-6Neu),
71.28 (C-3Gal), 70.53 (C-5Gal), 69.80 (C-2Gal), 69.28 (C-4Neu), 67.78
(C-8Neu), 67.28 (C-4Gal), 66.88 (C-7Neu), 62.54 (C-6GlcN), 62.21
(C-9Neu), 61.53 (C-6Gal), 55.17 (C-2GlcN), 53.12 (COOCH3), 49.06
(C-5Neu), 37.35 (C-3Neu), 23.14, 21.49, 20.89, 20.78, 20.74 × 3,
(C-6GlcN), 58.62 (C-2GlcN); MS (FAB) calcd for C21H28Cl3NO11
S
607.0, found m/z 607.1 [M+].
P h en yl O-(5-Aceta m id o-3,5-d id eoxy-D-glycer o-r-D-ga -
la cto-2-n on u lop yr a n osylon a te)-(2f3)-O-(â-D-ga la ctop y-
r a n osyl)-(1f4)-2-d eoxy-1-t h io-2-(2,2,2-t r ich lor oet h oxy-
ca r b on yla m in o)-â-D-glu cop yr a n osid e (7b ). The reaction
was performed in a total volume of 400 mL, and the following
reagents were added sequentially: 2.2 mM acceptor 6b (550
mg, 0.90 mmol), 50 mM (2-(N-morpholino)ethanesulfonic acid)
hydrate (MES, 40 mL of 0.5 M, pH 6.0), 100 mM MgCl2 (40
mL of 1 M), and 3.0 mM CMP-NeuNAc (60 mL of 20 mM).
The reaction was allowed to proceed at 37 °C after the addition
of the sialyltransferase (FUS-01/2, 150 units, 7.5 mL). The
reaction progress, i.e., the formation of material having Rf 0.15,
was monitored by TLC (MeOH-CHCl3-0.5% CaCl2 40:50:10).
After a total reaction time of 2 h, the crude product was
chromatographed (C18 reversed-phase column; elution with
water and then 65:35 water-MeOH) to yield pure trisaccha-
ride 7b (700 mg, 94%): [R]D -24.2 (c 0.1, H2O); 1H NMR (D2O)
δ 7.54-7.57 (m, 2 H, Ph), 7.39-7.44 (m, 3 H, Ph), 4.96 (d, 1
2430 J . Org. Chem., Vol. 68, No. 6, 2003

Simplifying Oligosaccharide Synthesis
20.62 × 2, 20.59 (10 × C, OdCCH3); MALDI-MS calcd for
mmol), and powdered molecular sieves 4 Å were dried at 40
°C in vacuo overnight. The mixture was dissolved in CH2Cl2
(2 mL) under an atmosphere of argon at 0 °C, and NIS (18
mg, 0.08 mmol) followed by TESOTf (6.75 µL, 0.03 mmol) were
added. After the mixture was stirred for 2 h, the reaction was
quenched with triethylamine and product was extracted with
CH2Cl2 (100 mL) three times. The combined organic layer was
washed with brine and water, dried over Na2SO4, and evapo-
rated. The residue was purified by silica gel chromatography
(ethyl acetate/methanol, 98:2) to yield pure 14 (50 mg, 80%):
1H NMR (CDCl3) δ 7.16-7.36 (m, 15 H, Ph), 5.53 (ddd, 1 H, J
) 9.0, 6.5, 2.5 Hz, H-8Neu), 5.40 (dd, 1 H, J ) 9.2, 3.0 Hz,
H-7Neu), 5.31 (t, 1 H, J ) 9.5 Hz, H-3GlcN), 5.08 (d, 2 H, J )
10.0 Hz, NHGlcN, NHNeu), 4.93 (dd, 1 H, J ) 10.5, 8.5 Hz, H-2Gal),
4.88 (d, 1 H, J ) 2.5 Hz, H-4Gal), 4.87 (td, 1 H, J ) 10.5, 5.0
Hz, H-4Neu), 4.86 (d, 1 H, J ) 12.5 Hz, 1/2 CH2CCl3), 4.81 (br,
1 H, J > 7.0 Hz, H-1GlcN), 4.76 (d, 1 H, J ) 11.5 Hz, 1/2 CH2-
Ph), 4.67 (d, 1 H, J ) 8.0 Hz, H-1Gal), 4.66 (s, 1 H, H-1Man),
4.60 (d, 1 H, J ) 14.0 Hz, 1/2 CH2Ph), 4.47-4.56 (m, 7 H, 2 ×
CH2Ph, H-3Gal, H-6GlcN, 1/2 CH2CCl3), 4.43 (dd, 1 H, J ) 12.5,
2.5 Hz, H-9Neu), 4.16 (dd, 1 H, J ) 12.0, 6.5 Hz, H-6GlcN), 4.11
C
51H65Cl3N2O28Na 1313.24, found m/z 1312.95 [M + Na+].
(4-O-Acet oxym et h yl)b en zyl O-(2,4,6-Tr i-O-a cet yl-â-D-
ga la ct op yr a n osyl)-(1f4)-O-[2-d eoxy-3,6-d i-O-a cet yl-2-
(2,2,2-tr ich lor oeth oxyca r bon yla m in o)-â-D-glu cop yr a n o-
syl]-(1f3)-4-O-a cet yl-2,6-d i-O-b en zoyl-â-D-ga la ct op yr a -
n osid e (12). The PEG-bound acceptor 10 (310 mg, 0.055
mmol), lactosaminyl donor 1b (70 mg, 0.083 mmol), and
powdered molecular sieves 4 Å were dried at 40 °C in vacuo
overnight. After being cooled to rt, the mixture was dissolved
in CH2Cl2 under an atmosphere of argon and NIS (37 mg, 0.165
mmol) followed by TESOTf (7.45 µL, 0.033 mmol) were added.
After the mixture was stirred for 2 h, the reaction was
quenched with N,N-diisopropylethylamine and the polymer
was precipitated with tert-butylmethyl ether (TBME, 150 mL).
The precipitate was recovered by filtration, after rinsing with
TBME followed by diethyl ether, and reprecipitated from
absolute EtOH (60 mL). The resulting solid was filtered,and
after washing with cold EtOH and ethyl ether it was dissolved
in CH2Cl2, filtered, and concentrated. The residue (320 mg)
containing 27% of PEGM bound trisaccharide 11 was dissolved
in CH2Cl2 (3 mL) and cooled in an ice bath under an
atmosphere of argon, and acetic anhydride (3 mL) followed by
Sc(OTf)3 (19 mg, 0.04 mmol) were added. The mixture was
warmed to rt and allowed to stir overnight. The polymer was
precipitated with TBME (100 mL) and filtered. The combined
filtrates were concentrated to dryness. The residue was
purified by preparative TLC (ethyl acetate-hexane 1:1) to
yield pure 12 (10.2 mg, 55%): [R]D +7.6 (c 0.3, CHCl3); 1H NMR
(CDCl3) δ 7.25-8.25 (m, 10 H, Bz), 7.10 (brs, 4 H, DOX), 5.53
(brd, 1 H, H-4I), 5.49 (dd, 1 H, J ) 10.0, 8.1 Hz, H-2I), 5.32
(brd, 1 H, H-4III), 5.06 (dd, 1 H, J ) 10.5, 7.8 Hz, H-2III), 5.01
(m, 2 H, NH, H-3II), 5.00 (s, 2 H, DOXCH2OAc), 4.94 (dd, 1 H,
J ) 10.5, 3.4 Hz, H-3III), 4.83 (d, 1 H, J ) 12.7 Hz, 1/2 OCH2-
DOX), 4.65 (d, 1 H, J ) 12.9, 9.0 Hz, H-6IIl), 4.61 (d, 1 H, J )
12.7 Hz, 1/2 OCH2DOX), 4.53 (d, 1 H, J ) 8.1 Hz, H-1I), 4.50
(m, 4 H, H-1II, H-1III, H-6I, 1/2 CH2CCl3), 4.39 (dd, 1 H, J )
11.6, 5.5 Hz, H-6I), 4.25 (d, 1 H, J ) 12.0 Hz, 1/2 CH2CCl3),
4.06 (brs, 2 H, 2 × H-6III), 3.99 (brt, H-5I), 3.94 (dd, 1 H, J )
12.7, 4.9 Hz, H-6II), 3.92 (dd, 1 H, J ) 10.0, 3.4 Hz, H-3I), 3.83
(brt, 1 H, H-5III), 3.73 (dd, 1 H, J ) 9.2, 9.2 Hz, H-4II), 3.43
(m, 2 H, H-2II, H-5II), 2.19, 2.12, 2.10, 2.04, 2.04, 2.02, 1.97,
1.95 (s, 24 H, OdCCH3); 13C NMR (CDCl3) δ 170.81, 170.44,
170.33, 170.10 × 2, 170.08 × 2, 169.08 (OdCCH3), 166.19,
164.82 (OdCPh), 153.71 (OdCOCH2CCl3), 136.66, 135.57,
133.51, 133.29, 129.79, 128.64, 128.47, 128.17, 128.10 (OdCPh,
DOX), 101.11 (C-1III), 101.08 (C-1II), 98.97 (C-1I), 95.40 (CCl3),
77.20 (C-3I), 75.80 (C-4II), 74.08 (CH2CCl3), 72.66 (C-5II), 71.40
(2 × C, C-3II, C-5I), 71.36 (2 × C, C-2I, C-5III), 70.89 (C-3III),
70.70 (C-5III), 69.66 (OCH2DOX), 69.46 (C-4I), 69.12 (C-2III),
66.59 (C-4III), 65.89 (DOXCH2OAc), 62.67 (C-6I), 60.75 (C-6III),
60.74 (C-6II), 56.35 (C-2II), 21.00, 20.84, 20.72, 20.63 × 2, 20.60
× 2, 20.49 (8 × C, OdCCH3); MS (FAB) calcd for C59H66Cl3-
NO28Na 1364.3, found m/z 1364.2 [M+].
(brs, 1 H, H-2Man), 3.98-4.04 (m, 4 H, H-5Neu, 2 × H-6Gal
,
H-9Neu), 3.89 (dd, 1 H, J ) 9.0, 2.5 Hz, H-3Man), 3.80-3.88 (m,
2 H, H-4Man, H-4GlcN), 3.84 (s, 3 H, COOCH3), 3.61-3.73 (m, 5
H, 2 × H-6Man, H-5Man, H-6Neu, H-5GlcN), 3.48 (dd, 1 H, J ) 12.5,
7.5 Hz, H-2GlcN), 3.32 (s, 3 H, OCH3), 2.72 (dd, 1 H, J ) 12.5,
4.5 Hz, H-3eqNeu), 2.23, 2.19, 2.16, 2.08, 2.06, 2.05, 2.04, 2.00,
1.98, 1.85 (s, 30 H, OdCCH3), 1.68 (t, 1 H, J ) 12.5 Hz,
H-3axNeu); 13C NMR (CDCl3) δ 170.87, 170.62, 170.55, 170.37,
170.31, 170.30, 170.21, 170.05, 169.61, 169.52 (10 × C,
OdCCH3), 167.94 (C-1Neu), 153.69 (O ) COCH2CCl3), 138.45,
138.20, 128.31, 128.30, 128.28, 127.94, 127.92, 127.63, 127.54,
127.44, 127.41 (18 × C, CH2Ph), 100.93 (C-1Gal), 99.30 (C-1GlcN),
98.45 (C-1Man), 96.75 (C-2Neu), 95.52 (CCl3), 78.19 (C-3Man),
76.43 (C-4GlcN), 74.93 (CH2CCl3), 74.55 (C-4Man), 74.30 (C-2Man),
73.16 (2 × C, CH2Ph), 72.90 (C-5GlcN), 72.05 (C-6Neu), 72.04
(C-3GlcN), 71.75 (C-5Man), 71.26 (C-3Gal), 71.25 (CH2Ph), 70.46
(C-5Gal), 69.88 (C-2Gal), 69.29 (2 × C, C-6Man, C-4Neu), 67.84
(C-8Neu), 67.30 (C-4Gal), 66.88 (C-7Neu), 62.64 (C-6GlcN), 62.16
(C-9Neu), 61.59 (C-6Gal), 56.35 (C-2GlcN), 54.76 (OCH3), 53.13
(COOCH3), 49.11 (C-5Neu), 37.38 (C-3Neu), 23.16, 21.50, 20.90,
20.82, 20.75 × 2, 20.69, 20.68, 20.67, 20.62 (10 × C, OdCCH3);
MALDI-MS calcd for C73H91Cl3N2O34Na 1669.46, found m/z
1669.01 [M + Na+].
Ack n ow led gm en t. We thank Ms. Marie-France
Karwaski for preparation of enzymes, Dr. J ean-Robert
Brisson and Ms. Suzon Larocque for NMR technical
help, and Mr. Ken Chan for MS analyses. We also
acknowledge the assistance of Ms. Tina. Dasgupta, a
Woman in Engineering and Science student, and Ms.
Kirsten Tisdall, a Chemistry Cooperative student, who
both worked on the early stages of this project. This is
NRC paper no. 42465.
Meth yl O-(Meth yl 5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-
3,5-d id e o x y -D -g ly c e r o -r-D -g a l a ct o-2-n o n u lo p y r a n o -
sylon a te)-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n o-
syl)-(1f4)-O-[2-d eoxy-3,6-d i-O-a cet yl-2-(2,2,2-t r ich lor o-
eth oxyca r bon yla m in o)-â-D-glu cop yr a n osyl]-(1f2)-3,4,6-
tr i-O-ben zyl-r-D-m an n opyr an oside (14). The mannose accep-
tor 13 (18 mg, 0.038 mmol), trisaccharide 2b (50 mg, 0.04
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedure and data for 8a ,b, 10, and 18. 1H and 13C NMR and
mass spectra of 1b, 5b,c, and 9. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O026569V
J . Org. Chem, Vol. 68, No. 6, 2003 2431