Chemoenzymatic synthesis of α-(1→3)-Gal(NAc)-terminating glycosides of complex tertiary sugar alcohols

Article abstract of DOI:10.1021/ja993004g

The scope of glycosyltransferases in the synthesis of oligosaccharide analogues has been expanded to include the use of "retaining" enzymes acting on complex tertiary C-branched disaccharide acceptors. The enzymes studied were α1,3-galactosyltransferase (

Full text of DOI:10.1021/ja993004g

J. Am. Chem. Soc. 1999, 121, 12063-12072
12063
Chemoenzymatic Synthesis of R-(1f3)-Gal(NAc)-Terminating
Glycosides of Complex Tertiary Sugar Alcohols
Xiangping Qian, Keiko Sujino, Albin Otter, Monica M. Palcic, and Ole Hindsgaul*
Contribution from the Department of Chemistry, UniVersity of Alberta,
Edmonton, Alberta T6G 2G2, Canada
ReceiVed August 18, 1999
Abstract: The scope of glycosyltransferases in the synthesis of oligosaccharide analogues has been expanded
to include the use of “retaining” enzymes acting on complex tertiary C-branched disaccharide acceptors. The
enzymes studied were R1,3-galactosyltransferase (R1,3-GalT, EC 2.4.1.151), which uses Galâ1f4Glcâ-OR
as the acceptor, and recombinant blood group A [R1,3-GalNAc transferase (GTA), EC 2.4.1.40] and B [R1,3-
Gal transferase (GTB), EC 2.4.1.37] glycosyltransferases, which use FucR1f2Galâ-OR as the acceptor in
their biosynthetic reactions. Chemical synthesis produced acceptor analogues in which the ring carbon bearing
the OH groups undergoing enzymatic glycosylation in the natural reactions had their C-H bond replaced with
a C-methyl or C-propyl bond. The tertiary C-methyl alcohols proved to be kinetically competent substrates
which permitted the enzymatic preparation of the expected product trisaccharides in near quantitative yields.
Introduction
Since structural information on protein (including enzyme)
binding sites is not generally available, chemical approaches
using engineered ligands are still necessary to study carbohy-
drate-protein interactions.7 Most of the ligand analogues used
have been acceptor analogues with hydroxyl groups selectively
deoxygenated, derivatized, or substituted with other functional
groups.6,8 Chemical mapping studies using these analogues help
elucidate hydrogen-bonding patterns and delineate the topology
of the binding site. However, there are only a few examples
known in which analogues having a C-bonded hydrogen atom
modified (termed C-branched sugars) were used.9 The scope
of chemical mapping on glycosyltransferases and enzymatic
synthesis has not yet included such sugar analogues. Probing
protein binding domains with C-branched analogues can provide
more information on the three-dimensional aspects of carbo-
Oligosaccharides on cell surfaces play an important role in
many biological recognition events including cell-cell adhesion,
bacterial attachment, and viral infection.1 The availability of
oligosaccharides and their analogues as inhibitors can provide
insight into their biological roles and might lead to the discovery
of novel carbohydrate-based therapeutics.2 Despite many ad-
vances in the chemical synthesis of oligosaccharides, it is still
time-consuming and often difficult to synthesize these highly
asymmetric and densely functionalized molecules.3 As well, no
replication system currently exists for amplifying minute
quantities of carbohydrates, nor is instrumentation commercially
available for the solid-phase synthesis of oligosaccharides.
Progress in recombinant DNA technology has made it
possible to express large quantities of almost any enzyme,
including glycosyltransferases that biosynthesize oligosaccha-
rides. These enzymes catalyze the transfer of a single pyranosyl
residue from a sugar nucleotide donor to a growing carbohydrate
chain. Many glycosyltransferases, isolated and cloned, are now
readily available in quantities sufficient for use in synthesis.4
The use of glycosyltransferases to prepare oligosaccharides
offers a number of advantages: high regio- and stereoselectivity,
no requirement for chemical protection and deprotection, and
very mild reaction conditions.5 Numerous examples have been
reported in which glycosyltransferases tolerate structural changes
on both donor and acceptor substrates, thus making enzymatic
synthesis a promising alternative for the preparation of both
natural and unnatural oligosaccharides.6
(5) For reviews on chemoenzymatic synthesis, see: (a) Wong, C.-H.;
Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Elsevier:
Oxford, 1994; pp 252-311. (b) Palcic, M. M. Methods Enzymol. 1994,
230, 300. (c) Crawley, S. C.; Palcic, M. M. In Modern Methods in
Carbohydrate Synthesis; Khan, S. H., O’Neil, R. A., Eds.; Harwood
Academic: Amsterdam, 1995; pp 492-517. (d) Wong, C.-H.; Halcomb,
R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34,
412. (e) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 521. (f) Gijsen, H. J. M.; Qiao, L.; Fitz,
W.; Wong, C.-H. Chem. ReV. 1996, 96, 443. (g) Takayama, S.; McGarvey,
G. J.; Wong, C.-H. Chem. Soc. ReV. 1997, 26, 407.
(6) (a) Palcic, M. M.; Hindsgaul, O. Trends Glycosci. Glycotechnol. 1996,
8, 37 and references therein. (b) Elhalabi, J. M.; Rice, K. G. Curr. Med.
Chem. 1999, 6, 93. (c) O¨ hrlein, R. Top. Curr. Chem. 1999, 200, 227.
(7) (a) Lemieux, R. U. Chem. Soc. ReV. 1989, 18, 347. (b) Lee, Y. C.;
Lee, R. T. Acc. Chem. Res. 1995, 28, 321. (c) Lemieux, R. U. Acc. Chem.
Res. 1996, 29, 373. (d) Solis, D.; Diaz-Maurino, T. In Glycosciences; Gabius,
H.-J., Gabius, S., Eds.; Chapman & Hall: Weinheim, 1997; pp 345-354.
(8) (a) Glaudemans, C. P. J. Chem. ReV. 1991, 91, 25. (b) Sierks, M. R.;
Bock, K.; Refn, S.; Svensson, B. Biochemistry 1992, 31, 8972. (c) Malet,
C.; Hindsgaul, O. J. Org. Chem. 1996, 61, 4649. (d) Lu, P.-P.; Hindsgaul,
O.; Li, H.; Palcic, M. M. Carbohydr. Res. 1997, 303, 283.
* To whom correspondence should be addressed. Tel.: (780) 492-4171.
Fax: (780) 492-7705. E-mail: Ole.Hindsgaul@ualberta.ca.
(1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Dwek, R. A. Chem. ReV.
1996, 96, 683.
(2) McAuliffe, J.; Hindsgaul, O. Chem. Ind. 1997, 170.
(3) For recent reviews on oligosaccharide synthesis, see: (a) Toshima,
K.; Tatsuta, K. Chem. ReV. 1993, 93, 1503. (b) Khan, S. H.; Hindsgaul, O.
In Molecular Glycobiology; Fukuda, M., Hindsgaul, O., Eds.; Oxford: New
York, 1994; pp 206-229. (c) Barresi, F.; Hindsgaul, O. J. Carbohydr. Chem.
1995, 14, 1043. (d) Boons, G.-J. Tetrahedron 1996, 52, 1095. (e) Schmidt,
R. R. In Glycosciences; Gabius, H.-J., Gabius, S., Eds.; Chapman & Hall:
Weinheim, 1997; pp 31-53.
(9) (a) Lindh, I.; Hindsgaul, O. J. Am. Chem. Soc. 1991, 113, 216. (b)
Hartmann, M.; Christian, R.; Zbiral, E. Monatsh. Chem. 1991, 122, 111.
(c) Palcic, M. M.; Skrydstrup, T.; Bock, K.; Le, N.; Lemieux, R. U.
Carbohydr. Res. 1993, 250, 87. (d) Sabesan, S.; Neira, S.; Davidson, F.;
Duus, J. O.; Bock, K. J. Am. Chem. Soc. 1994, 116, 1616. (e) Frandsen, T.
P.; Stoffer, B. B.; Palcic, M. M.; Hof, S.; Svensson, B. J. Mol. Biol. 1996,
263, 79. (f) Frandsen, T. P.; Palcic, M. M.; Dupont, C.; Svensson, B.
Carbohydr. Res. 1998, 314, 127.
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10.1021/ja993004g CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/10/1999

12064 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Qian et al.
Scheme 1
hydrate-protein interactions (Scheme 1), which complements
studies using analogues with modified hydroxyl groups. Func-
tional groups replacing hydrogens could, for example, serve as
molecular probes to explore which face of a given substrate is
making contact with the binding site and how close the contact
is (Scheme 1).
4-FucT) demonstrated that it is possible for mammalian gly-
cosyltransferases to catalyze the formation of glycosides of ter-
tiary alcohols.14 In light of this discovery, we decided to investi-
gate the scope of using mammalian glycosyltransferases to form
other types of glycosidic linkages with tertiary alcohol acceptors.
R1,3-Galactosyltransferase (R1,3-GalT, EC 2.4.1.151)15 and
human blood group A and B glycosyltransferases (GTA and
GTB, EC 2.4.1.40 and EC 2.4.1.37)16 were chosen for this study
because all three enzymes catalyze the formation of an R(axial)-
glycosidic linkage as does the R1,3/4-FucT. However, these
enzymes belong to a class of “retaining” enzymes which are
different from R1,3/4-FucT and other “inverting” glycosyltrans-
ferases. They transfer the sugar residue from the nucleotide
donor with overall retention, instead of inversion, of configu-
ration at the anomeric center. Chemical mapping studies with
the tertiary alcohol acceptor analogues will help us to understand
the molecular specificity of these glycosyltransferases and
increase the scope of enzymatic preparation of novel unnatural
oligosaccharides. The enzyme kinetic properties of these ana-
logues should also shed light on the molecular mechanisms of
R-glycosidic linkage formation by “retaining” glycosyltrans-
ferases.
Tertiary alcohols of C-branched sugar residues, in particular
those resulting from branching at the enzymatic reaction center,
will also be useful for probing the active sites of glycosyltrans-
ferases and for studying how steric hindrance affects the binding.
Since carbon-branched sugars are found only in plants and
microorganisms,10,11 it will be interesting to see whether
mammalian glycosyltransferases are able to recognize them.
Very few examples on chemical glycosylation of tertiary
alcohols have been reported since chemical glycosylation of such
hindered molecules is extremely difficult and frequently impos-
sible.12 Should glycosyltransferses be able to overcome the steric
limitations inherent in the glycosylation of the tertiary alcohols
of C-branched sugar residues, this enzymatic approach would
constitute a novel method for the preparation of glycosides of
complex tertiary alcohols. The resulting glycosides are expected
to have less flexible glycosidic linkages and would potentially
be useful in carbohydrate-protein recognition studies.9a,13
Our surprising finding of enzymatic fucosylation of a complex
tertiary alcohol by human milk R1,3/4-fucosyltransferase (R1,3/
In this paper, a series of C-alkyl-branched substrate analogues
were chemically synthesized and evaluated as acceptors for
R1,3-GalT, GTA, and GTB. Preparative enzymatic synthesis
of five R-(1f3)-Gal- or R-(1f3)-GalNAc-terminating glyco-
(10) (a) Watson, R. R.; Orenstein, N. S. AdV. Carbohydr. Chem. Biochem.
1975, 31, 135. (b) Grisebach, H. AdV. Carbohydr. Chem. Biochem. 1978,
35, 81. (c) Yoshimura, J. AdV. Carbohydr. Chem. Biochem. 1984, 42, 69.
(d) Lindberg, B. AdV. Carbohydr. Chem. Biochem. 1990, 48, 279.
(11) For some naturally occurring glycosides of tertiary alcohols, see:
(a) Kimura, Y.; Kobayashi, Y.; Takeda, T.; Ogihara, Y. J. Chem. Soc.,
Perkin Trans. 1981, 1923. (b) Chatterjee, D.; Bozic, C.; Aspinall, G. O.;
Brennan, P. J. J. Biol. Chem. 1988, 263, 4092. (c) Shimomura, H.; Sashida,
Y.; Mimaki, Y.; Adachi, T.; Yoshinari, K. Chem. Pharm. Bull. 1989, 37,
829. (d) Yamada, K.; Ojika, M.; Kigoshi, H. Angew. Chem., Int. Ed. 1998,
37, 1818.
(14) Qian, X.; Hindsgaul, O.; Li, H.; Palcic, M. M. J. Am. Chem. Soc.
1998, 120, 2184.
(15) (a) Blanken, W. M.; Van den Eijnden, D. H. J. Biol. Chem. 1985,
260, 12927. (b) Joziasse, D. H.; Shaper, N. L.; Salyer, L. S.; Van den
Eijnden, D. H.; van der Spoel, A. C.; Shaper, J. H. Eur. J. Biochem. 1990,
191, 75. (c) Henion, T. R.; Macher, B. A.; Anaraki, F.; Galili, U.
Glycobiology 1994, 4, 193. (d) Helland, A.-C.; Hindsgaul, O.; Palcic, M.
M.; Stults, C. L. M.; Macher, B. A. Carbohydr. Res. 1995, 276, 91. (e)
Nilsson, U. J.; Heerze, L. D.; Liu, Y.-C.; Armstrong, G. D.; Palcic, M. M.;
Hindsgaul, O. Bioconj. Chem. 1997, 8, 466. (f) Fang, J.; Li, J.; Chen, X.;
Zhang, Y.; Wang, J.; Guo Z,; Zhang, W.; Yu, L.; Brew, K.; Wang, P. G.
J. Am. Chem. Soc. 1998, 120, 6635. (g) Sujino, K.; Malet, M.; Hindsgaul,
O.; Palcic, M. M. Carbohydr. Res. 1998, 305, 483.
(12) (a) Beau, J.-M.; Jaurand, G.; Esnault, J.; Sinay¨, P. Tetrahedron Lett.
1987, 28, 1105. (b) Yan, L.; Kahne, D. Synlett 1995, 523. (c) Crich, D.;
Sun, S. J. Am. Chem. Soc. 1998, 120, 435.
(13) (a) Kolb, H. C.; Ernst, B. Chem. Eur. J. 1997, 3, 1571. (b)
Wilstermann, M.; Balogh, J.; Magnusson, G. J. Org. Chem. 1997, 62, 3659.
(c) Navarre, N.; van Oijen, A. H.; Boons, G. J. Tetrahedron Lett. 1997, 38,
2023. (d) Alibes, R.; Bundle, D. R. J. Org. Chem. 1998, 63, 6288. (e)
Bundle, D. R.; Alibes, R.; Nilar, S.; Otter, A.; Warwas, M.; Zhang, P. J.
Am. Chem. Soc. 1998, 120, 5317.
(16) (a) Yamamoto, F.-I.; Clausen, H.; White, T.; Marken, J.; Hakomori,
S.-I. Nature 1990, 345, 229. (b) Yamamoto, F.-I.; Hakomori, S.-I. J. Biol.
Chem. 1990, 265, 19257. (c) Seto, N. O. L.; Palcic, M. M.; Hindsgaul, O.;
Bundle, D. R.; Narang, S. A. Eur. J. Biochem. 1995, 234, 323. (d) Seto, N.
O. L.; Palcic, M. M.; Compston, C. A.; Li, H.; Bundle, D. R.; Narang, S.
A. J. Biol. Chem. 1997, 272, 14133.

Chemoenzymatic Synthesis of an Oligosaccharide Analogue
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 12065
Scheme 2a
a Reagents and conditions: (a) octanol, AgOTf, 4 Å MS, CH2Cl2, -50 to 0 °C, 4 h; (b) NaOMe, MeOH, 20 h, 37% (two steps); (c) (i) Bu2SnO,
benzene, reflux, 24 h, (ii) AllBr, Bu4NI, reflux, 14 h, 53%; (d) BnBr, NaH, DMF, 0 °C, 2 h, then room temperature, 14 h, 80%; (e) PdCl2, MeOH,
4 h, 83%; (f) Dess-Martin periodinane, CH2Cl2, 1.5 h, 72%; (g) 11, MeLi, THF, -78 to -40 °C, 1 h, 90%; 12, AllMgBr, THF, 0 °C, 4 h, 39%
12a, 51% 12b; (h) H2, 20% Pd(OH)2/C, MeOH, quantitative.
Scheme 3a
sides of complex tertiary sugar alcohols could thus be ac-
complished.
Results and Discussion
Chemical Synthesis. The general strategy for the preparation
of the required tertiary alcohol substrates involved the selective
protection of the parent disaccharide, permitting the oxidation
of the required ring carbon to the ketone. Nucleophilic addition
of the alkyl group from organometallic reagents followed by
deprotection gave the C-branched compounds. The products
were synthesized as their octyl glycosides, allowing the use of
a well-established radioactive “Sep-Pak assay” 17 for determining
kinetic parameters and simplifying the isolation of enzymatic
products by absorption onto reverse-phase (C18) cartridges.
As described in Scheme 2, the key step in the synthesis of
3′-C-branched lactosides 1 and 2 was the regioselective stan-
nylation of unprotected disaccharide 6, resulting in selective
3′-O-alkylation. Thus, octyl lactoside 6 was prepared by
glycosylation of bromide 5 with octanol followed by deacylation.
Regioselective allylation at O-3′ with dibutyltin oxide and allyl
bromide gave 7 in 53% yield.18 Benzylation, followed by
removal of the allyl group with PdCl2,19 provided 9 with OH-
3′ free. Oxidation of 9 was achieved with Dess-Martin
periodinane20 to provide 10 in 72% yield. Nucleophilic addition
of methyllithium to ketone 10 gave exclusively the product of
axial attack (11) in excellent yield (95%), while the reaction of
allylmagnesium bromide with 10 provided a mixture of both
epimers (12a and 12b) in almost a 1:1 ratio. Hydrogenolysis
with Pd(OH)2/C gave unprotected 3′-C-branched lactosides 1
and 2. NOE studies employing one-dimensional TROESY
experiments21 showed that there are significant NOEs between
a Reagents and conditions: (a) Dess-Martin periodinane, CH2Cl2,
2 h, 61%; (b) 15, MeLi, THF, -78 to -40 °C, 1.5 h, 55%; 16, AllMgBr,
THF, 0 °C, 2 h, 33% 16a, 29% 16b; (c) H2, 20% Pd(OH)2/C, MeOH,
95% (for 3), 94% (for 4a), 92% (for 4b).
the 3′-C-methyl protons in 1 (3′-C-CH2CH2CH3 protons in 2a)
and H-1 of the Gal residue, while there are no NOEs between
the 3′-C-propyl protons and H-1′ in 2b.
Analogues 3 and 4 with carbon-branching at the 3-position
of FucR1f2Gal were prepared from the known precursor 13,22
following the same oxidation-nucleophilic addition sequence
(Scheme 3). Disaccharide 13 was oxidized with Dess-Martin
periodinane to provide 14 (61%). Nucleophilic attack of MeLi
(17) Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycocon-
jugate J. 1988, 5, 49.
(18) (a) Alais, J.; Maranduba, A.; Veyrieres, A. Tetrahedron Lett. 1983,
24, 2383. (b) David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.
(19) Ogawa, T.; Yamamoto, H. Agric. Biol. Chem. 1985, 49, 475.
(20) (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4156. (b)
Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(21) (a) Hwang, T.-L.; Shaka, A. J. J. Magn. Reson. Ser. B 1993, 102,
155. (b) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz,
R. W. J. Am. Chem. Soc. 1984, 106, 811. (c) Kessler, H.; Mronga, S.;
Gemmecker, G. Magn. Reson. Chem. 1991, 29, 527. (d) Geen, H.; Freeman,
R. J. Magn. Reson. 1991, 93, 93.
(22) Lowary, T. L.; Hindsgaul, O. Carbohydr. Res. 1994, 251, 33.

12066 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Qian et al.
Table 1
R
analogue
R1,3-GaITa
analogue
GTBb
GTAc
H
17
Km ) 1.5 ( 0.1 mM
Vmax ) 100%
Km ) 23 ( 5 mM
Vmax ) 38%
18
Km ) 76 ( 13 µM
Vmax ) 100%
Km ) 17 ( 2 µM
Vmax ) 100%
CH3
Pr
1
3
Km ) 209 ( 15 µM
Vmax ) 60%
Km ) 39 ( 3 µM
Vmax ) 64%
2a
inactive
4a
Km ) 2200 ( 200 µM
Vmax ) 8%
Km ) 2800 ( 300 µM
Vmax ) 26%
a For assay conditions, see ref 15g. b Reactions were incubated at 37 °C for 30 min in 20 µL total volume with 35 mM sodium cacodylate buffer
(pH 7.0), 20 mM MnCl2, 1 mg/mL BSA, 200 µM UDP-Gal, UDP-[6-3H]Gal (about 75 000 dpm), 20 µU GTB. c Reactions were incubated at 37
°C for 20 min in 20 µL total volume with 35 mM sodium cacodylate buffer (pH 7.0), 20 mM MnCl2, 1 mg/mL BSA, 70 µM UDP-GalNAc,
UDP-[6-3H]GalNAc (about 75 000 dpm), 9 µU GTA.
on the carbonyl group of 14 provided the axial attack product
C-methyl 15 in 61% yield, while reaction of allylmagnesium
bromide with 15 again afforded epimers 16a and 16b in a 1:1
ratio. Hydrogenolysis of 15 and 16 gave the deprotected 3-C-
branched disaccharides 3 and 4. The configurations at the
branching carbons were assigned on the basis of observed strong
NOEs between H-1 of Gal and the 3-C-methyl protons in 3
(3-C-CH2CH2CH3 protons in 4a) and strong NOEs between H-2
of Gal and 3-C-CH2CH2CH3 protons in 4b.
Enzymatic Assays. R1,3-GalT, which is absent in humans,
catalyzes the formation of GalR1f3Gal epitopes, the major
xenoactive antigen which causes hyperacute rejection during
xenotransplantation.23 The enzyme can use both Galâ1f
4GlcNAcâ-OR and Galâ1f4Glcâ-OR as acceptors.15 Acceptor
hydroxyl group mapping studies15g showed that Gal OH-4 is,
according to the terminology of Lemieux,24 a key polar group
essential for substrate recognition. Substitution of Gal OH-3
with an amino group produces an inhibitor for this enzyme.15d
These results suggested that OH-3′ is not essential for binding
to the enzyme, although it is the site of glycosylation. Surpris-
ingly, the carbon-branched analogue 3′-C-methyl lactoside 1
was found to be a substrate in the radioactive Sep-Pak assay.
The Km for 1 was 23 mM, about 15 times higher than that for
the parent n-octyl lactoside 17 (Km ) 1.5 mM). The maximal
rate of transfer (Vmax) decreased to 38% of that of 17 (Table 1).
However, replacement of the hydrogen with a bulkier propyl
group produced an inactive compound 2a when tested as an
acceptor at a concentration of 2 mM in a 6-h incubation and as
an inhibitor at a concentration of 5 mM.
GTB are highly homologous glycosyltransferases and differ by
only four amino acid residues.16a Human GTB exhibits about
40% sequence identity with R1,3-GalT.26 It would thus be
reasonable to expect that both GTA and GTB could also tolerate
carbon-branching at the 3-position of the Gal residue. In fact,
both 3-C-methyl- and 3-C-propyl-branched analogues, 3 and
4a, were found to be acceptors for these two enzymes. As shown
in Table 1, replacement of the hydrogen atom at the site of the
enzymatic reaction with a methyl group did not significantly
influence the Km and Vmax values. However, substitution with a
larger propyl group dramatically altered the kinetic properties.
The Km values were almost 30 and 165 times higher and Vmax
decreased to 8% and 26%, respectively.
It is quite remarkable that all three enzymes can tolerate the
introduction of large substituents at the carbon bearing the
hydroxyl group that undergoes glycosylation, although it is not
required for the recognition. However, as revealed by kinetic
data, the steric increase does make it more difficult for the
enzymes to bind and transfer. The larger the alkyl group, the
higher the Km and the lower the Vmax values. Among the three
enzymes, the introduction of an alkyl substituent has more
impact on Km and Vmax for R1,3-GalT than for GTA and GTB
since the Vmax/Km value for 1 was only 2.5% of that for 17
with R1,3-GalT, while the Vmax/Km value for 3 was only de-
creased to 22-28% of that for 1825 with GTA and GTB. This
is also in agreement with the observation that no detectable
transfers to the 3′-C-propyl-branched analogue 2a was observed
with R1,3-GalT, while 4a still displayed activity with GTA and
GTB. The three enzymes seem to have similar molecular
specificity with respect to the â-Gal residue: OH-4 is required
for binding while OH-3 is not essential and C-methyl-branching
is tolerated at C-3 of the â-Gal residue. The similar molecular
specificity and the homology of these enzymes indicate that
they may have similar three-dimensional structure at the active
sites.
As mentioned earlier, GTA and GTB are similar to R1,3-
GalT and catalyze the formation of R1f3 galactosidic linkage
with retention of configuration. GTA catalyzes the transfer of
GalNAc from UDP-GalNAc to the blood group (O)H precursor
structure FucR1f2Galâ-OR to give the blood group A trisac-
charide GalNAcR1f3[FucR1f2]Galâ-OR. GTB uses the same
acceptor substrate but catalyzes the transfer of Gal from UDP-
Gal to produce the blood group B trisaccharide GalR1f3-
[FucR1f2]Galâ-OR. Systematic studies have revealed that Gal
OH-3 is not essential for recognition by both enzymes, as
observed in the case of R1,3-GalT.22,25 Furthermore, GTA and
A two-step double-displacement mechanism involving the
formation and breakdown of glycosyl-enzyme intermediate is
most likely involved for the retaining glycosyltransferases25 as
well as for glycosidases.27 Since a direct SN2-type substitution
is unlikely to occur in the second step for the very hindered
tertiary alcohols investigated here, we propose that the break-
(23) (a) Galili, U. Immunol. Today 1993, 14, 480. (b) Butler, D. Nature
1998, 391, 320.
(24) Lemieux, R. U. Proceedings of the VIIIth International Symposium
on Medicinal Chemistry; Swedish Parmaceutical Press: Stockholm, 1985;
pp 329-351.
(25) Lowary, T. L.; Hindsgaul, O. Carbohydr. Res. 1993, 249, 163.
(26) Breton, C.; Bettler, E.; Joziasse, D. H.; Geremia, R. A.; Imberty,
A. J. Biochem. 1998, 123, 1000.

Chemoenzymatic Synthesis of an Oligosaccharide Analogue
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 12067
Scheme 4
Scheme 5
down of the glycosyl-enzyme intermediate begins prior to the
nucleophilic attack by OH. The process would thus have
significant SN1 character (Scheme 4), as suggested for R1,3-
FucT V28 and R-glucosyltransferase.29 It is interesting that the
four enzymes, R1,3/4-FucT,14 R1,3-GalT, GTA, and GTB,
which all catalyze the formation of an R(axial)-glycosidic
linkage with either retention or inversion of configuration,
tolerate C-branching at the glycosylation site.
Preparative Synthesis and Characterization of Enzymatic
Products. To be certain that the analogues found to be acceptors
in radioactive enzymatic assays were indeed glycosylated in the
expected manner, preparative reactions of 1, 3, and 4a using
the corresponding enzymes were performed. Mass spectrometry
and extensive NMR studies were employed to confirm the
structures of the enzymatic products.
Trisaccharide 19 was obtained in 90% yield from the reaction
of 1 with UDP-Gal catalyzed by R1,3-GalT (Scheme 5). As
expected, protons of the 3-C-methyl group were shifted down-
field from 1.24 to 1.37 ppm.30 The 13C chemical shift of the
quaternary branching carbon (C-3′) in 19 also shifted downfield
from 74.7 to 80.9 ppm with no significant chemical shift change
in other carbon resonances. Moreover, to exclude the possibility
of incorrect linkage formation (1f4 instead of 1f3) in 19,
trisaccharide 20 was prepared in 95% yield with the use of R1,4-
galactosyltransferase31 (R1,4-GalT, Scheme 5). This precaution
was necessary in view of the conformational peculiarities of
GalR1f3Gal linkages, which result in much stronger nuclear
Overhauser enhancements between RGal-H1 and âGal-H4 com-
pared to RGal-H1:âGal-H3.32,33 As can be seen in Figure 1,
trisaccharide 19 shows the expected H1′′/3′-C-methyl NOE as
well as an even stronger H1′′/H4′ interaction, which is consistent
with earlier observations in GalR1f3Galâ1f4Glc32 and the
A and B trisaccharides,33 whereas the 1f4-linked trisaccharide
20 shows the H1′′/H4′ interaction only. These NOE results,
together with the RGal-H1:âGal-C3 correlation in HMBC34
studies of 19, prove unambiguously the 1f3 linkage of 19.
As shown in Scheme 6, the A and B trisaccharide analogues
21-24 were obtained in excellent yields using GTA and GTB
with UDP-GalNAc or UDP-Gal as donors. Complete conversion
of the poor substrate 4a was achieved by increasing the amounts
of enzyme and donor and the reaction time. Detailed NMR
studies revealed the NOE pattern characteristic for the A and B
trisaccharides:33 a stronger NOE from H-1 (R-Gal or R-GalNAc)
to H-4 (â-Gal) than from H-1 (R-Gal or R-GalNAc) to protons
of alkyl substituents at C-3. Significant downfield shifts for the
(27) (a) Sinnott, M. L. Chem. ReV. 1990, 90, 1171. (b) Kempton, J. B.;
Withers, S. G. Biochemistry 1992, 31, 9961. (c) McCarter, J. D.; Withers,
S. G. J. Am. Chem. Soc. 1996, 118, 241. (d) Heightman, T. D.; Vasella, A.
T. Angew. Chem., Int. Ed. 1999, 38, 750.
(28) (a) Murray, B. W.; Takayama, S.; Schultz, J.; Wong, C.-H.
Biochemistry 1996, 35, 11183. (b) Murray, B. W.; Wittmann, V.; Burkart,
M. D.; Hung S.-C.; Wong, C.-H. Biochemistry 1997, 36, 823.
(29) Kakinuma, H.; Yuasa, H.; Hashimoto, H. Carbohydr. Res. 1998,
312, 103.
(30) Wong, C.-H.; Ichikawa, Y.; Krach, T.; Narvor, C. G.-L.; Dumas,
D. P.; Look, G. C. J. Am. Chem. Soc. 1991, 113, 8137.
(31) Wakarchuk, W. W.; Cunningham A.; Watson, D. C.; Young, N.
M. Protein Eng. 1998, 11, 295.
(32) Li, J.; Ksebati, M. B.; Zhang, W.; Guo, Z.; Wang, J.; Yu, L.; Fang,
J.; Wang, P. G. Carbohydr. Res. 1999, 315, 76.
(33) Otter, A.; Lemieux, R. U., Ball, R. G.; Venot, A. P.; Hindsgaul,
O.; Bundle, D. R. Eur. J. Biochem. 1999, 259, 295.
(34) Bax, A.; Marion, D. J. Magn. Reson. 1988, 78, 186.

12068 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Qian et al.
Figure 1. One-dimensional 600-MHz spectra of trisaccharides 19 and 20 (bottom) together with 1D-TROESY spectra obtained from selective
excitation of the H-1′′ resonance (top).
Scheme 6
Conclusions
In summary, efficient chemical routes were developed leading
to axially tertiary C-branched alcohols of disaccharide acceptors
for three related “retaining” R1,3-glycosyltransferases. Despite
the potential complexity of the mechanisms of these enzymes,
they all tolerated the replacement of a C-H bond by a C-CH3
bond directly at the site of glycosylations, permitting the
preparative synthesis and characterization of the expected
product trisaccharides. Larger C-propyl derivatives were not
preparatively useful. The trisaccharides produced by this
chemoenzymatic approach are analogues of naturally occurring
carbohydrate antigens and should find application in the study
of carbohydrate-protein recognition. In particular, such C-
methyl compounds should reveal the steric requirements of the
binding sites of antibodies, lectins (selectins), glycosyltrans-
ferases, and glycosidases near the OH group where the substitu-
tion has occurred. Of potentially greater interest is that the
C-branched oligosaccharides should be conformationally more
restricted due to the increased steric interaction between sugar
rings and may thus be useful for evaluating entropic contribu-
tions to protein-oligosaccharide binding.
Experimental Section
protons (∼0.15 ppm) of 3-C-methyl or 3-C-CH2CH2CH3 and
for the carbon resonance C-3 (∼7 ppm) were also observed,
compared to the starting disaccharides. In addition, conforma-
tion-independent proof for correct linkages in trisaccharides 21-
24 was obtained by HMBC experiments34 through 13C/1H
correlations across the glycosidic linkage.
Unless otherwise noted, column chromatography was performed on
Silica Gel 60 (E. Merck, 40-63 µm, Darmstadt). Iatrobeads (beaded
silica gel 6RS-8060) were from Iatron Laboratories (Tokyo). Millex-
GV (0.22 µm) filter units were from Millipore (Missisauga, ON,
Canada), and C18 Sep-Pak sample preparation cartridges were from

Chemoenzymatic Synthesis of an Oligosaccharide Analogue
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 12069
1
Waters (Missisauga, ON, Canada). H NMR spectra were recorded at
5.93 (dddd, 1H, J ) 17.0, 10.5, 5.5, 5.5 Hz), 5.32 (dddd, 1H, J )
17.0, 1.5, 1.5, 1.5 Hz), 5.18 (dddd, 1H, J ) 10.5, 1.5, 1.5, 1.5 Hz),
5.02 (d, 1H, J ) 10.8 Hz), 4.97 (d, 1H, J ) 11.5 Hz), 4.92 (d, 1H, J
) 10.8 Hz), 4.84-4.71 (m, 4H), 4.56 (d, 1H, J ) 11.5 Hz), 4.54 (d,
1H, J ) 12.0 Hz), 4.45 (d, 1H, J ) 7.7 Hz), 4.44 (d, 1H, J ) 10.8
Hz), 4.38 (d, 1H, J ) 8.3 Hz), 4.34 (d, 1H, J ) 11.8 Hz), 4.24 (d, 1H,
J ) 11.8 Hz), 4.17 (m, 2H), 3.99-3.86 (m, 3H), 3.83-3.67 (m, 3H),
3.61-3.48 (m, 3H), 3.45-3.28 (m, 5H), 1.64 (m, 2H), 1.46-1.22 (m,
10H), 0.89 (t, 3H, J ) 7.0 Hz); 13C NMR (75 MHz, CDCl3) δ 139.3,
139.2, 139.0, 138.8, 138.6, 138.2, 135.1, 128.4, 128.3, 128.2, 128.1,
128.0 (2 × C), 127.9 (2 × C), 127.8, 127.7, 127.6, 127.5, 127.4 (2 ×
C), 127.1, 116.4, 103.7, 102.8, 83.1, 82.5, 81.9, 79.6, 77.0, 75.4, 75.0,
74.7, 73.6, 73.5, 73.1, 73.0, 71.5, 70.1, 68.5, 68.2, 31.9, 29.8, 29.5,
29.3, 26.2, 22.7, 14.1; HR-ESMS calcd for C65H78O11Na (M + Na+)
1057.5442, found 1057.5435.
300 MHz (Varian Inova 300), 360 MHz (Bruker AM 360), 500 MHz
(Varian Unity 500), or 600 MHz (Varian Inova 600). 13C NMR spectra
were recorded at 75 MHz (Bruker AM 300) or 125 MHz (Varian Unity
500). Chemical shifts were referenced to solvent peaks for solutions
in CDCl3, CD2Cl2, and CD3OD, or external 1% acetone (1H, δ 2.225;
13C, δ 31.07) for solutions in D2O. High-resolution electrospray mass
spectra were recorded on a Micro-mass ZabSpec Hydroid Sector-TOF.
R1,3-GalT was isolated from calf thymus.15e GTA and GTB were
expressed and cloned in E. coli.16c,d R1,4-GalT was a generous gift
from Dr. Warren W. Wakarchuk.31 Alkaline phosphatase (calf intestine)
was from Boehringer Mannheim. Enzymatic assays were performed
according to a well-established radioactive Sep-Pak assay method.17,22,25
Kinetic parameters, Km and Vmax, were derived from the best fit of the
Michaelis-Menten equation using nonlinear regression analysis with
the SigmaPlot 4.1 program. The maximal velocity (Vmax) was arbitrarily
set to 100% for the parent acceptors 17 and 18.
Octyl â-D-Galactopyranosyl-(1f4)-â-D-glucopyranoside (6). Bro-
mide 5 (2 g, 1.76 mmol) and ground 4Å molecular sieves in CH2Cl2
(20 mL) were stirred under argon at room temperature for 20 min and
cooled to -50 °C. Octanol (0.45 mL, 2.82 mmol) and silver triflate
(725 mg, 2.82 mmol) were added. The reaction mixture was stirred
for 4 h while it was allowed to warm to 0 °C. After neutralization with
Et3N, the mixture was filtered through Celite, washed with CH2Cl2,
and concentrated. The partially purified disaccharide (obtained by
column chromatography using 3:1 hexanes/EtOAc) was dissolved in
MeOH, and a freshly prepared methanolic solution of NaOMe was
added. The mixture was stirred at room temperature for 27 h, neutralized
with Amberlite IR-120 (H+), filtered, concentrated, and recrystallized
from MeOH to yield 6 (295 mg, 37% for two steps) as a white solid:
1H NMR (500 MHz, D2O) δ 4.48 (d, 1H, J ) 8.0 Hz), 4.45 (d, 1H, J
) 8.0 Hz), 3.98 (dd, 1H, J ) 12.1, 2.1 Hz), 3.94-3.88 (m, 2H), 3.82-
3.75 (m, 3H), 3.74-3.62 (m, 5H), 3.60-3.56 (m, 1H), 3.54 (dd, 1H,
J ) 10.0, 7.8 Hz), 3.30 (m, 1H), 1.62 (m, 2H), 1.39-1.23 (m, 10H),
0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (125 MHz, D2O) δ 103.8, 102.8,
79.3, 76.2, 75.6, 75.3, 73.7, 73.4, 71.8, 71.6, 69.4, 61.8, 61.0, 31.9,
29.5, 29.2, 29.1, 25.8, 22.8, 14.2; HR-ESMS calcd for C20H38O11Na
(M + Na+) 477.2311, found 477.2315.
Octyl 3-O-Allyl-â-D-galactopyranosyl-(1f4)-â-D-glucopyranoside
(7). A suspension of 6 (454 mg, 1 mmol) in benzene (25 mL) was
refluxed for 24 h in the presence of Bu2SnO (300 mg, 1.2 mmol) in a
flask equipped with a Dean-Stark separator. Allyl bromide (1.6 mL,
18.5 mmol) and tetrabutylammonium iodide (185 mg, 0.5 mmol) were
added. The resulting solution was refluxed for 14 h. After evaporation
of solvent, the residue was dissolved in hot MeOH. On cooling, a
crystalline solid was obtained on filtration, and the filtrate was
concentrated. Purification on a column of Iatrobeads (10:1 CH2Cl2/
MeOH) yielded 7 (260 mg, 53%) as a white solid: 1H NMR (360 MHz,
CD3OD) δ 5.98 (dddd, 1H, J ) 17.0, 10.5, 5.5, 5.5 Hz), 5.32 (dddd,
1H, J ) 17.0, 1.5, 1.5, 1.5 Hz), 5.15 (dddd, 1H, J ) 10.5, 1.5, 1.5, 1.5
Hz), 4.37 (d, 1H, J ) 7.8 Hz), 4.27 (d, 1H, J ) 7.8 Hz), 4.22 (dddd,
1H, J ) 13.0, 5.5, 1.5, 1.5 Hz), 4.12 (dddd, 1H, J ) 13.0, 5.5, 1.5, 1.5
Hz), 3.99 (dd, 1H, J ) 3.0, 0.5 Hz), 3.92-3.81 (m, 3H), 3.77 (dd, 1H,
J ) 11.4, 7.4 Hz), 3.69 (dd, 1H, J ) 11.4, 4.7 Hz), 3.62 (dd, 1H, J )
9.7, 7.8 Hz), 3.59-3.47 (m, 4H), 3.38 (m, 1H), 3.32 (m, 1H), 3.23
(dd, 1H, J ) 8.8, 7.8 Hz), 1.61 (m, 2H), 1.43-1.23 (m, 10H), 0.89 (t,
3H, J ) 7.0 Hz); 13C NMR (125 MHz, CD3OD) δ 136.5, 117.4, 105.1,
104.3, 82.2, 80.9, 76.9, 76.5, 75.4, 74.8, 71.8, 71.7, 71.0, 67.1, 62.5,
62.1, 33.0, 30.8, 30.6, 30.4, 27.1, 23.7, 14.4; HR-ESMS calcd for
C23H42O11Na (M + Na+) 517.2625, found 517.2625.
Octyl 3-O-Allyl-2,4,6-tri-O-benzyl-â-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-â-D-glucopyranoside (8). A solution of 7 (240 mg,
0.49 mmol) and benzyl bromide (0.5 mL, 3.92 mmol) in DMF (5 mL)
was cooled to 0 °C. Sodium hydride (196 mg, 60% dispension in
mineral oil, 4.9 mmol) was added, and the mixture was stirred at 0 °C
for 2 h. The reaction was then slowly warmed to room temperature
and stirred overnight. After quenching with MeOH, the mixture was
extracted with CH2Cl2. The organic layer was washed with water and
brine and then dried (Na2SO4), filtered, and concentrated. The residue
was purified by column chromatography (6:1 hexanes/EtOAc) to yield
8 (400 mg, 80%): 1H NMR (360 MHz, CDCl3) δ 7.50-7.10 (m, 30H),
Octyl 2,4,6-Tri-O-benzyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-
O-benzyl-â-D-glucopyranoside (9). To a solution of 8 (400 mg, 0.39
mmol) in MeOH (15 mL) was added PdCl2 (34 mg, 0.19 mmol). The
mixture was stirred at room temperature for 4 h. After evaporation of
MeOH, the residue was purified by column chromatography (4:1
hexanes/EtOAc) to yield 9 (320 mg, 83%) as a syrup: 1H NMR (360
MHz, CDCl3) δ 7.50-7.10 (m, 30H), 5.06 (d, 1H, J ) 10.8 Hz), 4.96
(d, 1H, J ) 10.8 Hz), 4.86 (d, 1H, J ) 11.5 Hz), 4.85-4.76 (m, 3H),
4.75 (d, 1H, J ) 11.5 Hz), 4.69 (d, 1H, J ) 11.2 Hz), 4.63 (d, 1H, J
) 11.5 Hz), 4.52-4.39 (m, 4H), 4.32 (d, 1H, J ) 11.8 Hz), 4.05-
3.94 (m, 2H), 3.91-3.76 (m, 3H), 3.66-3.51 (m, 5H), 3.51-3.39 (m,
4H), 1.69 (m, 2H), 1.50-1.22 (m, 10H), 0.92 (t, 3H, J ) 7.0 Hz); 13
C
NMR (75 MHz, CDCl3) δ 139.2, 138.8 (2 × C), 138.5, 138.4, 138.1,
128.5, 128.4, 128.3, 128.1 (2 × C), 128.0, 127.9, 127.8, 127.7, 127.6
(2 × C), 127.5, 127.1, 103.7, 102.7, 82.9, 81.8, 80.7, 76.8, 76.0, 75.3,
75.2, 75.1, 75.0, 74.2, 73.4, 73.3, 73.2, 70.1, 68.4, 68.0, 31.9, 29.8,
29.5, 29.3, 26.2, 22.7, 14.1; HR-ESMS calcd for C62H74O11Na (M +
Na+) 1017.5129, found 1017.5137.
Octyl 2,4,6-Tri-O-benzyl-â-D-xylo-hex-3-ulopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-â-D-glucopyranoside (10). A solution of Dess-
Martin periodinane (190 mg, 0.45 mmol) in CH2Cl2 (6 mL) was added
to a stirred solution of 9 (300 mg, 0.30 mmol) in CH2Cl2 (7 mL), and
stirring was continued for 1.5 h. The mixture was poured into a saturated
aqueous NaHCO3 solution containing Na2S2O3 and extracted with
EtOAc. The organic layer was washed with water and brine, dried (Na2-
SO4), filtered, concentrated, and submitted to column chromatography
(5:1 hexanes/EtOAc) to yield 10 (215 mg, 72%) as a solid: 1H NMR
(300 MHz, CDCl3) δ 7.40-7.10 (m, 30H), 4.93 (d, 1H, J ) 10.4 Hz),
4.89 (d, 1H, J ) 10.4 Hz), 4.79 (d, 1H, J ) 11.0 Hz), 4.69 (d, 1H, J
) 11.0 Hz), 4.64 (d, 1H, J ) 7.6 Hz), 4.63 (d, 1H, J ) 11.5 Hz), 4.48
(d, 1H, J ) 12.0 Hz), 4.46 (d, 1H, J ) 12.0 Hz), 4.44, (d, 1H, J )
11.0 Hz), 4.40 (d, 1H, J ) 12.0 Hz), 4.37 (d, 1H, J ) 8.3 Hz), 4.34 (d,
1H, J ) 12.0 Hz), 4.31 (d, 1H, J ) 11.0 Hz), 4.30 (d, 1H, J ) 7.6
Hz), 4.20 (d, 1H, J ) 12.0 Hz), 3.96-3.82 (m, 3H), 3.80-3.70 (m,
2H), 3.68-3.46 (m, 3H), 3.44-3.32 (m, 4H), 1.62 (m, 2H), 1.44-
1.20 (m, 10H), 0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (75 MHz, CDCl3)
δ 204.3, 139.1, 138.7, 138.4, 138.1, 137.4, 137.0, 128.5, 128.4 (2 ×
C), 128.3, 128.2, 128.1 (3 × C), 128.0, 127.8, 127.6 (2 × C), 127.5,
127.2, 103.7, 103.6, 83.3, 83.1, 82.0, 81.0, 77.7, 75.3, 75.0, 74.9, 73.5,
73.3, 73.2, 73.1, 72.5, 70.1, 68.6, 66.8, 31.9, 29.8, 29.5, 29.3, 26.2,
22.7, 14.1; HR-ESMS calcd for C62H72O11Na (M + Na+) 1015.4972,
found 1015.4972.
Octyl 2,4,6-Tri-O-benzyl-3-C-methyl-â-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-â-D-glucopyranoside (11). Compound 10 (85 mg,
0.09 mmol) was dissolved in THF (1.5 mL) and cooled under argon to
-78 °C. Methyllithium (1.4 M, 0.1 mL) in diethyl ether was added
with stirring. The reaction mixture was stirred for 1 h while it was
allowed to warm to -40 °C. The reaction was poured into saturated
NH4Cl and extracted with EtOAc. The organic layer was washed with
water and brine, dried (Na2SO4), filtered, concentrated, and submitted
to column chromatography (5:1 hexanes/EtOAc) to yield 11 (78 mg,
90%): 1H NMR (300 MHz, CDCl3) δ 7.40-7.10 (m, 30H), 5.01 (d,
1H, J ) 11.0 Hz), 4.89 (d, 1H, J ) 11.0 Hz), 4.77 (d, 1H, J ) 11.6
Hz), 4.76 (d, 1H, J ) 11.0 Hz), 4.71 (d, 1H, J ) 11.0 Hz), 4.67 (d,
1H, J ) 11.6 Hz), 4.64 (d, 1H, J ) 11.7 Hz), 4.60 (d, 1H, J ) 11.7
Hz), 4.53 (d, 1H, J ) 8.0 Hz), 4.46 (d, 1H, J ) 12.3 Hz), 4.42 (d, 1H,

12070 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Qian et al.
J ) 12.3 Hz), 4.37 (d, 1H, J ) 11.8 Hz), 4.34 (d, 1H, J ) 7.7 Hz),
4.28 (d, 1H, J ) 11.8 Hz), 3.97-3.86 (m, 2H), 3.74 (dd, 1H, J )
11.0, 4.2 Hz), 3.67 (dd, 1H, J ) 11.0, 1.5 Hz), 3.64-3.46 (m, 4H),
3.44-3.28 (m, 5H), 1.62 (m, 2H), 1.44-1.18 (m, 10H), 1.14 (s, 3H),
0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (75 MHz, CDCl3) δ 139.4, 139.0,
138.8, 138.5, 138.3, 137.9, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0,
127.9 (2 × C), 127.8, 127.7, 127.6, 127.4 (2 × C), 127.2, 127.1, 103.7,
101.9, 83.1 (2 × C), 82.1, 81.9, 77.3, 75.7, 75.2 (2 × C), 75.1, 75.0,
74.9, 73.5, 72.9, 71.9, 70.1, 68.5, 68.3, 31.9, 29.8, 29.5, 29.3, 26.2,
22.7, 19.2, 14.1; HR-ESMS calcd for C63H76O11Na (M + Na+)
1031.5285, found 1031.5297.
Octyl 2,4,6-Tri-O-benzyl-3-C-allyl-â-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-â-D-glucopyranoside (12a) and Octyl 2,4,6-Tri-
O-benzyl-3-C-allyl-â-D-gulopyranosyl-(1f4)-2,3,6-tri-O-benzyl-â-D-
glucopyranoside (12b). To a solution of 10 (40 mg, 0.04 mmol) in
THF (3 mL) at 0 °C was added allylmagnesium bromide (1.0 M, 0.1
mL) in diethyl ether with stirring under argon. The reaction mixture
was stirred at 0 °C for 4 h, quenched with saturated NH4Cl, and
extracted with EtOAc. The organic layer was washed with water and
brine, dried (Na2SO4), filtered, concentrated, and submitted to column
chromatography (20:1 toluene/EtOAc) to yield 12a (16 mg, 39%) and
12b (21 mg, 51%).
For 12a: 1H NMR (300 MHz, CD2Cl2) δ 7.40-7.20 (m, 30H), 5.86
(dddd, 1H, J ) 17.0, 10.0, 9.6, 4.8 Hz), 5.06 (ddd, 1H, J ) 10.0, 1.5,
1.5 Hz), 5.02 (d, 1H, J ) 11.0 Hz), 4.88 (d, 1H, J ) 11.0 Hz), 4.84
(ddd, 1H, J ) 17.0, 1.5, 1.5 Hz), 4.78-4.62 (m, 7H), 4.53 (d, 1H, J )
12.3 Hz), 4.45 (d, 1H, J ) 12.0 Hz), 4.43 (d, 1H, J ) 12.0 Hz), 4.36
(d, 1H, J ) 8.0 Hz, H-1), 4.33 (d, 1H, J ) 12.3 Hz), 3.94 (t, 1H, J )
9.4 Hz), 3.90 (dt, 1H, J ) 9.6, 6.5 Hz), 3.82 (dd, 1H, J ) 10.8, 3.8
Hz), 3.68 (dd, 1H, J ) 10.8, 1.4 Hz), 3.64-3.39 (m, 7H), 3.38-3.28
(m, 2H), 2.76 (dddd, 1H, J ) 15.0, 4.8, 1.5, 1.5 Hz), 2.44 (d, 1H, J )
1.5 Hz), 1.97 (bdd, 1H, J ) 15.0, 9.6 Hz), 1.64 (m, 2H), 1.44-1.22
(m, 10H), 0.88 (t, 3H, J ) 7.0 Hz); 13C NMR (75 MHz, CD2Cl2) δ
139.9, 139.4, 139.0, 138.8, 138.7, 133.8, 128.8, 128.7, 128.6, 128.5,
128.4, 128.3 (2 × C), 128.2, 128.1 (2 × C), 128.0, 127.9, 127.8 (2 ×
C), 127.7, 127.4, 127.3, 118.3, 104.1, 101.8, 83.7, 83.2, 82.1, 78.7,
77.3, 76.4, 75.9, 75.8, 75.3, 75.2, 75.0, 73.6, 73.1, 71.7, 70.3, 68.8,
68.7, 36.3, 32.2, 30.2, 29.8, 29.7, 26.6, 23.1, 14.3; HR-ESMS calcd
for C65H78O11Na (M + Na+) 1057.5442, found 1057.5440.
1.62 (m, 2H), 1.38-1.24 (m, 10H), 1.24 (s, 3H), 0.86 (t, 3H, J ) 7.0
Hz); 13C NMR (125 MHz, D2O) δ 102.8, 102.7, 79.4, 75.5, 75.4, 75.3,
74.7, 74.1, 74.0, 73.6, 71.6, 62.2, 61.0, 31.9, 29.5, 29.2, 29.1, 25.8,
22.8, 18.6, 14.2; HR-ESMS calcd for C21H41O11 (M + H+) 469.2649,
found 469.2648.
Octyl 3-C-Propyl-â-D-galactopyranosyl-(1f4)-â-D-glucopyrano-
side (2a) and Octyl 3-C-Propyl-â-D-gulopyranosyl-(1f4)-â-D-glu-
copyranoside (2b). Compounds 12a (7 mg, 6.8 µmol) and 12b (9 mg,
8.7 µmol) were treated in the same fashion as for 1 to give, respectively,
2a (3.3 mg) and 2b (4.1 mg) in quantitative yields.
For 2a: 1H NMR (600 MHz, D2O) δ 4.58 (d, 1H, J ) 8.2 Hz), 4.47
(d, 1H, J ) 7.9 Hz), 3.96 (dd, 1H, J ) 12.3, 2.2 Hz), 3.91 (dt, 1H, J
) 10.0, 7.0 Hz), 3.85 (dd, 1H, J ) 7.4, 4.8 Hz), 3.82 (bs, 1H), 3.78
(dd, 1H, J ) 12.3, 5.0 Hz), 3.75 (dd, 1H, J ) 11.8, 7.4 Hz), 3.73 (dd,
1H, J ) 11.8, 4.8 Hz), 3.67 (dt, 1H, J ) 10.0, 7.0 Hz), 3.65-3.60 (m,
3H), 3.57 (m, 1H), 3.30 (m, 1H), 1.73 (m, 1H), 1.62 (m, 2H), 1.56 (m,
1H), 1.41 (m, 2H), 1.38-1.22 (m, 10 H), 0.92 (t, 3H, J ) 7.0 Hz),
0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (125 MHz, D2O) δ 102.8, 102.5,
79.4, 76.3, 75.5, 75.4, 74.9, 74.6, 73.6, 71.6, 70.0, 62.2, 61.0, 33.0,
31.9, 29.5, 29.2, 29.1, 25.8, 22.8, 15.9, 14.8, 14.2; HR-ESMS calcd
for C23H44O11Na (M + Na+) 519.2781, found 519.2786.
For 2b: 1H NMR (600 MHz, D2O) δ 4.68 (d, 1H, J ) 8.0 Hz), 4.47
(d, 1H, J ) 8.0 Hz), 4.06 (ddd, 1H, J ) 8.0, 4.1, 0.8 Hz), 3.97 (dd,
1H, J ) 12.2, 4.2 Hz), 3.91 (dt, 1H, J ) 10.0, 7.0 Hz), 3.81 (dd, 1H,
J ) 12.2, 5.1 Hz), 3.75 (dd, 1H, J ) 12.0, 8.0 Hz), 3.73 (dd, 1H, J )
12.0, 4.1 Hz), 3.67 (dt, 1H, J ) 10.0, 7.0 Hz), 3.65-3.60 (m, 3H),
3,57 (m, 1H), 3.38 (d, 1H, J ) 8.0 Hz), 3.30 (m, 1H), 1.76 (m, 1H),
1.66-1.60 (m, 3H), 1.40-1.22 (m, 12H), 0.92 (t, 3H, J ) 7.0 Hz),
0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (125 MHz, D2O) δ 102.9, 102.6,
79.9, 76.6, 75.5, 75.4 (2 × C), 73.7, 72.8, 71.6, 69.6, 62.2, 61.1, 36.4,
31.9, 29.5, 29.2, 29.1, 25.8, 22.8, 15.4, 14.6, 14.2; HR-ESMS calcd
for C23H44O11Na (M + Na+) 519.2781, found 519.2786.
Octyl 2,3,4-Tri-O-benzyl-r-L-fucopyranosyl-(1f2)-4,6-di-O-ben-
zyl-â-D-xylo-hexopyranosid-3-ulose (14). Compound 13 (135 mg, 0.15
mmol) was oxidized in the same fashion as for 10. The crude product
was purified by column chromatography (24:1 toluene/EtOAc) to yield
14 (81 mg, 61%): 1H NMR (500 MHz, CDCl3) δ 7.50-7.20 (m, 25H),
5.05 (d, 1H, J ) 3.7 Hz), 4.99 (d, 1H, J ) 11.5 Hz), 4.98 (d, 1H, J )
11.5 Hz), 4.92 (d, 1H, J ) 11.6 Hz), 4.73 (d, 1H, J ) 11.6 Hz), 4.70
(d, 1H, J ) 7.9 Hz), 4.69 (d, 1H, J ) 11.5 Hz), 4.64 (d, 1H, J ) 11.6
Hz), 4.60 (d, 1H, 7.9 Hz), 4.54 (d, 1H, J ) 11.8 Hz), 4.52 (d, 1H, J )
11.8 Hz), 4.46 (d, 1H, J ) 11.8 Hz), 4.41 (d, 1H, J ) 11.8 Hz), 4.21
(bq, 1H, J ) 6.6 Hz), 4.10 (dd, 1H, J ) 10.2, 3.5 Hz), 3.96 (dd, 1H,
J ) 10.2, 2.9 Hz), 3.90 (dt, 1H, J ) 9.4, 6.4 Hz), 3.87 (d, 1H, J ) 1.4
Hz), 3.78-3.72 (m, 2H), 3.70 (m, 1H), 3.61 (bd, 1H, J ) 2.9 Hz),
3.42 (dt, 1H, J ) 9.4, 6.4 Hz), 1.52 (m, 2H), 1.30-1.18 (m, 10H),
1.08 (d, 3H, J ) 6.6 Hz), 0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (125
MHz, CDCl3) δ 203.4, 139.2, 138.8, 138.5, 137.9, 136.6, 128.7, 128.6,
128.5 (2 × C), 128.4, 128.3 (3 × C), 128.2, 127.8, 127.7, 127.6, 127.5,
127.4 (2 × C), 102.6, 96.0, 80.5, 79.1, 78.3, 77.6, 76.1, 74.9, 73.8,
73.7, 73.5, 72.6 (2 × C), 70.1, 67.8, 66.8, 31.9, 29.7, 29.5, 29.4, 26.2,
22.7, 16.5, 14.1; HR-ESMS calcd for C55H66O10Na (M + Na+)
909.4554, found 909.4547.
Octyl 2,3,4-Tri-O-benzyl-r-L-fucopyranosyl-(1f2)-4,6-di-O-ben-
zyl-3-C-methyl-â-D-galactopyranoside (15). Compound 14 (50 mg,
56 µmol) was treated with MeLi in the same fashion as for 11. The
crude product was purified by column chromatography (25:1 toluene/
EtOAc) to yield 15 (28 mg, 55%): 1H NMR (500 MHz, CDCl3) δ
7.40-7.20 (m, 25H), 5.33 (d, 1H, J ) 3.5 Hz), 4.94 (d, 1H, J ) 11.6
Hz), 4.82 (d, 1H, J ) 11.6 Hz), 4.77 (d, 1H, J ) 11.6 Hz), 4.74 (d,
1H, J ) 11.6 Hz), 4.72 (d, 1H, J ) 11.6 Hz), 4.70 (d, 1H, J ) 11.6
Hz), 4.62 (d, 1H, J ) 11.6 Hz), 4.61 (d, 1H, J ) 11.6 Hz), 4.48 (d,
1H, J ) 11.6 Hz), 4.44 (d, 1H, J ) 11.6 Hz), 4.26 (d, 1H, J ) 7.9
Hz), 4.16 (bq, 1H, J ) 6.5 Hz), 4.02 (dd, 1H, J ) 10.1, 3.5 Hz), 3.96
(dd, 1H, J ) 10.1, 2.7 Hz), 3.82-3.76 (m, 2H), 3.74 (d, 1H, J ) 7.9
Hz), 3.62 (dd, 1H, J ) 2.7, 1.0 Hz), 3.60-3.57 (m, 2H), 3.41 (d, 1H,
J ) 0.9 Hz), 3.35 (dt, 1H, J ) 9.3, 7.0 Hz), 1.53 (m, 2H), 1.28-1.19
(m, 13H), 1.08 (d, 3H, J ) 6.5 Hz), 0.85 (t, 3H, J ) 7.0 Hz); 13C
NMR (125 MHz, CDCl3) δ 139.0, 138.9, 138.4, 138.2, 138.0, 129.0,
128.7, 128.5, 128.4 (2 × C), 128.3 (2 × C), 128.2 (3 × C), 128.1,
127.9 (2 × C), 127.8 (2 × C), 127.7, 127.5 (2 × C), 101.5, 98.7, 81.8,
For 12b: 1H NMR (300 MHz, CD2Cl2) δ 7.40-7.10 (m, 30H), 5.97
(dddd, 1H, J ) 17.0, 10.0, 6.4, 3.7 Hz), 5.18-5.08 (m, 2H), 4.88 (d,
1H, J ) 11.0 Hz), 4.87 (d, 1H, J ) 7.9 Hz), 4.86 (d, 1H, J ) 11.0
Hz), 4.71 (d, 1H, J ) 11.0 Hz), 4.69 (d, 1H, J ) 11.0 Hz), 4.60 (d,
1H, J ) 11.0 Hz), 4.59 (d, 1H, J ) 11.0 Hz), 4.50-4.45 (m, 3H), 4.37
(d, 1H, J ) 12.0 Hz), 4.36 (d, 1H, J ) 7.8 Hz), 4.13 (dd, 1H, J ) 8.3,
6.2 Hz), 3.96 (t, 1H, J ) 8.5 Hz), 3.90 (dt, 1H, J ) 9.5, 6.5 Hz), 3.77
(dd, 1H, J ) 10.8, 4.0 Hz), 3.72 (dd, 1H, J ) 10.8, 2.0 Hz), 3.62-
3.43 (m, 5H), 3.40-3.33 (m, 2H), 3.30 (dd, 1H, J ) 9.2, 7.8 Hz), 2.62
(dddd, 1H, J ) 14.5, 6.4, 1.5, 1.5 Hz), 2.54 (bdd, 1H, J ) 14.5, 7.7
Hz), 2.18 (s, 1H), 1.64 (m, 2H), 1.44-1.20 (m, 10H), 0.89 (t, 3H, J )
7.0 Hz); 13C NMR (75 MHz, CD2Cl2) δ 139.9, 139.5, 139.1, 138.8 (2
× C), 138.6, 134.2, 128.7 (2 × C), 128.6 (2 × C), 128.5 (2 × C),
128.4, 128.2 (3 × C), 128.0, 127.9, 127.8 (2 × C), 127.7 (2 × C),
127.4, 118.8, 104.0, 101.5, 83.1, 81.9, 80.6, 77.6, 76.7, 75.8, 75.4 (2
× C), 75.1, 75.0, 73.7, 73.5, 72.3, 70.3, 68.8, 68.6, 39.8, 32.3, 30.2,
29.8, 29.7, 26.6, 23.1, 14.3; HR-ESMS calcd for C65H78O11Na (M +
Na+) 1057.5442, found 1057.5450.
Octyl 3-C-Methyl-â-D-galactopyranosyl-(1f4)-â-D-glucopyrano-
side (1). The benzyl-protected 11 (21 mg, 21 µmol) was dissolved in
MeOH (10 mL), and the solution was stirred under a stream of H2 in
the presence of 20% Pd(OH)2/C (25 mg) for 10 h. The catalyst was
removed by filtration through a Millex-GV 0.22-µm filter and the
solvent evaporated. The product was purified by redissolution in water
and then passing the solution through a Waters C18 Sep-Pak cartridge.
The cartridge was washed with water and eluted with gradient MeOH-
H2O 1:4 to 2:1. The elulant containing the compound was concentrated,
redissolved in water, filtered through a Millex-GV 0.22-µm filter, and
lyophilized to yield 1 (9.5 mg, quant) as a white solid: 1H NMR (500
MHz, D2O) δ 4.52 (d, 1H, J ) 8.1 Hz), 4.47 (d, 1H, J ) 8.0 Hz), 3.96
(dd, 1H, J ) 12.2, 2.2 Hz), 3.94-3.87 (m, 2H), 3.81-3.72 (m, 3H),
3.67 (dt, 1H, J ) 10.0, 7.0 Hz), 3.65-3.55 (m, 5H), 3.30 (m, 1H),

Chemoenzymatic Synthesis of an Oligosaccharide Analogue
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 12071
79.8, 79.4, 78.0, 77.2, 75.8, 75.7, 74.8, 73.7, 73.6, 72.9, 72.7, 70.1,
69.5, 66.8, 31.9, 29.8, 29.5, 29.3, 26.1, 22.7, 20.3, 16.7, 14.1; HR-
ESMS calcd for C56H70O10Na (M + Na+) 925.4867, found 925.4872.
Octyl r-L-Fucopyranosyl-(1f2)-3-C-methyl-â-D-galactopyrano-
side (3). The benzyl-protected 15 (13 mg, 14 µmol) was treated in the
same fashion as for 1 to give 3 (6 mg, 95%): 1H NMR (600 MHz,
D2O) δ 5.35 (d, 1H, J ) 4.0 Hz), 4.53 (d, 1H, J ) 8.1 Hz), 4.37 (bq,
1H, J ) 6.6 Hz), 3.91 (dt, 1H, J ) 9.6, 6.6 Hz), 3.89 (dd, 1H, J )
10.5, 3.4 Hz), 3.84-3.77 (m, 3H), 3.76 (dd, 1H, J ) 11.5, 7.2 Hz),
3.72 (dd, 1H, J ) 11.5, 3.8 Hz), 3.66 (d, 1H, J ) 8.1 Hz), 3.64
(dt, 1H, J ) 9.6, 6.6 Hz), 3.55 (bs, 1H), 1.60 (m, 2H), 1.36-1.24 (m,
13H), 1.21 (d, 1H, J ) 6.6 Hz), 0.86 (t, 3H, J ) 7.0 Hz); 13C NMR
(125 MHz, D2O) δ 102.6, 99.5, 78.3, 76.3, 74.8, 74.5, 72.8, 71.5, 70.3,
69.4, 67.5, 62.1, 31.9, 29.8, 29.3, 29.2, 26.2, 22.8, 19.3, 16.2, 14.2;
HR-ESMS calcd for C21H40O10Na (M + Na+) 475.2519, found
475.2526.
Octyl r-L-Fucopyranosyl-(1f2)-3-C-propyl-â-D-galactopyrano-
side (4a) and Octyl r-L-Fucopyranosyl-(1f2)-3-C-propyl-â-D-
gulopyranoside (4b). Compound 14 (20 mg, 22 µmol) was treated
with allylmagnesium bromide as described for the preparation of 12.
The crude product was purified by column chromatography (8:1
hexanes/EtOAc) to yield 16a (7 mg, 33%) and 16b (6 mg, 29%).
Hydrogenolysis of 16a (7 mg, 7.5 µmol) and 16b (4 mg, 4.3 µmol) as
described for 1 gave 4a (3.4 mg, 94%) and 4b (1.9 mg, 92%).
For 4a: 1H NMR (600 MHz, D2O) δ 5.32 (d, 1H, J ) 4.0 Hz, H-1′),
4.60 (d, 1H, J ) 8.2 Hz, H-1), 4.36 (bq, 1H, J ) 6.6 Hz), 3.91 (dt, J
) 9.9, 6.6 Hz), 3.89 (dd, 1H, J ) 10.4, 3.3 Hz), 3.81-3.73 (m, 5H),
3.72 (dd, 1H, J ) 11.3, 5.0 Hz), 3.69 (d, 1H, J ) 8.2 Hz), 3.65 (dd,
1H, J ) 9.9, 6.6 Hz), 1.92 (m, 1H), 1.67-1.58 (m, 3H), 1.42 (m, 1H),
1.37-1.24 (m, 11H), 1.22 (d, 3H, J ) 6.6 Hz), 0.93 (t, 3H, J ) 7.0
Hz), 0.87 (t, 3H, J ) 7.0 Hz); HR-ESMS calcd for C23H44O10Na (M +
Na+) 503.2832, found 503.2838.
4.28 (bt, 1H, J ) 6.5 Hz), 4.02 (bd, 1H, J ) 3.3 Hz), 3.99-3.79 (m,
9H), 3.73-3.60 (m, 6H), 3.57 (m, 1H), 3.29 (m, 1H), 1.62 (m, 2H),
1.39-1.24 (m, 13H), 0.86 (t, 3H, J ) 7.0 Hz); 13C NMR (125 MHz,
D2O) δ 103.1, 102.8, 101.8, 84.0, 80.0, 75.7, 75.5, 75.4, 74.7, 74.3,
73.7, 72.4, 71.6, 69.8 (2 × C), 69.3, 61.6, 61.5, 61.0, 31.9, 29.5, 29.2,
29.1, 25.8, 22.8, 19.3, 14.2; HR-ESMS calcd for C27H50O16Na (M +
Na+) 653.2997, found 653.3002.
Octyl r-D-Galactopyranosyl-(1f3)-[r-L-fucopyranosyl-(1f2)]-
3-C-methyl-â-D-galactopyranoside (21). The reaction mixture con-
tained disaccharide 3 (2.4 mg, 5.3 µmol), UDP-Gal (5.0 mg, 8.2 µmol),
750 mU of blood group B transferase (in 150 µL containing 35 mM
sodium cacodylate, 20 mM MnCl2, and 1 mg/mL BSA), 40 µL of
concentrated buffer (350 mM sodium cacodylate, 200 mM MnCl2, and
10 mg/mL BSA, pH 7.0), and 4 µL of alkaline phosphatase (1 U/µL).
The reaction was incubated with rotation at room temperature for 2
days. The mixture was purified as described for 19 to yield 21 (3.2
mg, quantitative) as a white solid: 1H NMR (600 MHz, D2O) δ 5.38
(d, 1H, J ) 4.0 Hz), 5.32 (d, 1H, J ) 4.0 Hz), 4.60 (d, 1H, J ) 8.0
Hz), 4.46 (bq, 1H, J ) 6.6 Hz), 4.14 (m, 1H), 4.03 (d, 1H, J ) 3.1
Hz), 3.98 (s, 1H), 3.93 (dt, 1H, J ) 9.8, 6.6 Hz), 3.90-3.69 (m, 11H),
3.65 (dt, 1H, J ) 9.8, 6.6 Hz), 1.60 (m, 2H), 1.48 (s, 3H), 1.37-1.24
(m, 10H), 1.22 (d, 3H, J ) 6.6 Hz), 0.87 (t, 3H, J ) 7.0 Hz); 13C
NMR (125 MHz, D2O) δ 101.1, 99.3, 93.3, 83.8, 76.4, 74.3, 72.8, 72.6,
71.5, 70.6, 70.3, 70.2, 69.9, 69.1, 68.8, 67.5, 62.2, 61.9, 31.9, 29.8,
29.4, 29.2, 26.3, 22.8, 17.2, 16.2, 14.2; HR-ESMS calcd for C27H50O15-
Na (M + Na+) 637.3047, found 637.3057.
Octyl r-D-2-Acetamido-2-deoxygalactopyranosyl-(1f3)-[r-L-fu-
copyranosyl-(1f2)]-3-C-methyl-â-D-galactopyranoside (22). The
reaction mixture contained disaccharide 3 (1.2 mg, 2.7 µmol), UDP-
GalNAc (5.4 mg, 8.3 µmol), 1.45 mU of blood group A transferase
(in 490 µL containing 35 mM sodium cacodylate, 20 mM MnCl2, and
1 mg/mL BSA), 80 µL of concentrated buffer (350 mM sodium
cacodylate, 200 mM MnCl2, and 10 mg/mL BSA, pH 7.0), and 10 µL
of alkaline phosphatase (1 U/µL). The reaction was incubated with
rotation at 37 °C for 4 days, and the reaction mixture was purified as
described for 19 to yield 22 (1.6 mg, 92%) as a white solid: 1H NMR
(600 MHz, D2O) δ 5.36 (d, 1H, J ) 3.8 Hz), 5.32 (d, 1H, J ) 3.7 Hz),
4.60 (d, 1H, J ) 8.2 Hz), 4.50 (bq, 1H, J ) 6.6 Hz), 4.22-4.17 (m,
2H), 4.06 (d, 1H, J ) 2.4 Hz), 4.00 (s, 1H), 3.94 (dt, 1H, J ) 9.8, 6.6
Hz), 3.90-3.84 (m, 2H), 3.84-3.70 (m, 8H), 3.66 (dt, 1H, J ) 9.8,
6.6 Hz), 2.02 (s, 3H), 1.49 (s, 3H), 1.61 (m, 2H), 1.38-1.26 (m, 10H),
1.24 (d, 3H, J ) 6.6 Hz), 0.87 (t, 3H, J ) 7.0 Hz); 13C NMR (125
MHz, D2O) δ 175.5, 101.1, 99.1, 92.7, 83.9, 76.2, 75.1, 72.8, 72.0,
71.6, 70.3, 69.8, 69.2, 69.0 (2 × C), 67.6, 62.2, 61.7, 50.7, 31.9, 29.8,
29.4, 29.2, 26.3, 22.9, 22.8, 18.9, 16.2, 14.2; HR-ESMS calcd for
C29H53NO15Na (M + Na+) 678.3313, found 678.3310.
Octyl r-D-Galactopyranosyl-(1f3)-[r-L-fucopyranosyl-(1f2)]-
3-C-propyl-â-D-galactopyranoside (23). The reaction mixture con-
tained disaccharide 4a (1.2 mg, 2.5 µmol), UDP-Gal (3.1 mg, 5.1 µmol),
0.5 U of blood group B transferase (in 100 µL containing 35 mM
sodium cacodylate, 20 mM MnCl2, and 1 mg/mL BSA), 35 µL of
concentrated buffer (350 mM sodium cacodylate, 200 mM MnCl2, and
10 mg/mL BSA, pH 7.0), 5 µL of alkaline phosphatase (1 U/µL), and
200 µL of water. The reaction was incubated with rotation at 37 °C
for 22 days. Additional UDP-Gal (6.2 mg), B transferase (1.5 U), and
alkaline phosphatase (10 µL) were added during the incubation period.
The reaction mixture was purified as described for 19 to yield 23 (1.6
mg, quantitative) as a white solid: 1H NMR (600 MHz, D2O) δ 5.43
(d, 1H, J ) 4.2 Hz), 5.29 (d, 1H, J ) 4.0 Hz), 4.68 (d, 1H, J ) 7.9
Hz), 4.44 (bq, 1H, J ) 6.6 Hz), 4.19-4.16 (m, 2H), 4.06 (d, 1H, J )
3.3 Hz), 3.92 (dt, 1H, J ) 9.9, 6.6 Hz), 3.91-3.86 (m, 3H), 3.84-
3.69 (m, 8H), 3.66 (dt, 1H, J ) 9.9, 6.6 Hz), 2.02 (m, 1H), 1.77 (m,
1H), 1.66-1.56 (m, 3H), 1.40-1.24 (m, 11H), 1.22 (d, 3H, J ) 6.6
Hz), 0.90 (t, 3H, J ) 7.0 Hz), 0.87 (t, 3H, J ) 7.0 Hz); 13C NMR (125
MHz, D2O) δ 101.3, 99.5, 93.5, 84.9, 77.5, 74.1, 72.8, 72.4, 71.5, 70.4,
70.3, 69.8, 69.2, 69.1, 67.6 (2 × C), 62.1, 61.4, 34.9, 31.9, 29.8, 29.4,
29.2, 26.3, 22.8, 16.7, 16.2, 15.2, 14.2; HR-ESMS calcd for C29H54O15-
Na (M + Na+) 665.3360, found 665.3355.
For 4b: 1H NMR (600 MHz, D2O) δ 5.01 (d, 1H, J ) 3.8 Hz), 4.77
(d, 1H, J ) 8.0 Hz), 4.38 (bq, 1H, J ) 6.6 Hz), 4.00 (dd, J ) 7.2, 5.1
Hz), 3.89 (dd, 1H, J ) 10.5, 3.3 Hz), 3.86 (m, 1H), 3.83-3.79 (m,
2H), 3.76 (dd, 1H, J ) 11.7, 7.5 Hz), 3.73-3.68 (m, 2H), 3.65 (bs,
1H), 3.42 (d, 1H, J ) 8.0 Hz), 1.92 (m, 1H), 1.72-1.60 (m, 3H), 1.42-
1.26 (m, 12H), 1.20 (d, 3H, J ) 6.6 Hz), 0.93 (t, 3H, J ) 7.0 Hz),
0.86 (t, 3H, J ) 7.0 Hz); HR-ESMS calcd for C23H44O10Na (M + Na+)
503.2832, found 503.2838.
Octyl r-D-Galactopyranosyl-(1f3)-â-D-3-C-methylgalactopyran-
osyl-(1f4)-â-D-glucopyranoside (19). Disaccharide 1 (3.4 mg, 7.3
µmol), UDP-Gal (8.9 mg, 14.6 µmol), and 10 µL of alkaline
phosphatase (1 U/µL) were added to a 0.5-mL solution of calf thymus
R1,3-GalT (120 mU in 30 mM sodium cacodylate, 20 mM MnCl2,
0.1% Triton X-100, pH 6.5). The mixture was incubated with rotation
at room temperature for 5 days. The reaction mixture was filtered
through glass wool and loaded onto two sequential C18 Sep-Pak
cartridges. The cartridges were washed with water (100 mL) and 10%
aqueous methanol (40 mL) and then eluted with 50% aqueous MeOH
(40 mL). The 50% eluant was concentrated, redissolved in water, filtered
through a Millex-GV 0.22-µm filter, and lyophilized to yield 19 (4.1
mg, 90%) as a white solid: 1H NMR (600 MHz, D2O) δ 5.35 (d, 1H,
J ) 4.0 Hz), 4.58 (d, 1H, J ) 8.2 Hz), 4.48 (d, 1H, J ) 8.0 Hz), 4.23
(m, 1H), 4.00-3.96 (m, 2H), 3.94 (dd, 1H, J ) 10.2, 3.3 Hz), 3.92-
3.87 (m, 3H), 3.86 (dd, 1H, J ) 10.2, 4.0 Hz), 3.81-3.70 (m, 6H),
3.68 (dt, 1H, J ) 10.0, 6.8 Hz), 3.64-3.59 (m, 2H), 3.58 (m, 1H),
3.30 (m, 1H), 1.63 (m, 2H), 1.38-1.24 (m, 13H), 0.86 (t, 3H, J ) 7.0
Hz); 13C NMR (125 MHz, D2O) δ 102.8, 102.4, 93.1, 80.9, 79.7, 75.5,
75.4, 74.9, 73.6, 73.3, 71.9, 71.6, 71.4, 70.2, 70.0, 69.0, 62.2, 61.9,
61.1, 31.9, 29.5, 29.2, 29.1, 25.8, 22.8, 16.0, 14.2; HR-ESMS calcd
for C27H50O16Na (M + Na+) 653.2997, found 653.2998.
Octyl r-D-Galactopyranosyl-(1f4)-â-D-3-C-methylgalactopyran-
osyl-(1f4)-â-D-glucopyranoside (20). The reaction mixture contained
disaccharide 1 (1.8 mg, 3.8 µmol), UDP-Gal (5.0 mg, 8.2 µmol), 20
mU of R1,4-GalT (in 200 µL containing 52 mM Hepes, 10 mM
ammonium acetate, pH 7.5), and 10 µL of alkaline phosphatase (1
U/µL). The reaction was incubated with rotation at room temperature
for 4 days. The mixture was purified as described for 19 to yield 20
(2.3 mg, 95%) as a while solid: 1H NMR (600 MHz, D2O) δ 5.01 (d,
1H, J ) 4.0 Hz), 4.60 (d, 1H, J ) 8.0 Hz), 4.47 (d, 1H, J ) 8.0 Hz),
Octyl r-D-2-Acetamido-2-deoxygalactopyranosyl-(1f3)-[r-L-fu-
copyranosyl-(1f2)]-3-C-propyl-â-D-galactopyranoside (24). The
reaction mixture contained disaccharide 4a (1.2 mg, 2.5 µmol), UDP-

12072 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Qian et al.
GalNAc (3.3 mg, 5.1 µmol), 1.4 U of blood group A transferase (in
200 µL containing 35 mM sodium cacodylate, 20 mM MnCl2, and 1
mg/mL BSA), 10 µL of concentrated buffer (350 mM sodium
cacodylate, 200 mM MnCl2, and 10 mg/mL BSA, pH 7.0), 10 µL of
alkaline phosphatase (1 U/µL), and 220 µL of water. The reaction was
incubated with rotation at 37 °C for 26 days. Additional UDP-GalNAc
(9.3 mg), A transferase (4.5 U), and alkaline phosphatase (10 µL) were
added during the incubation period. The reaction mixture was purified
as described for 19 to yield 24 (1.6 mg, 94%) as a white solid: 1H
NMR (600 MHz, D2O) δ 5.36 (d, 1H, J ) 4.0 Hz), 5.32 (d, 1H, J )
4.0 Hz), 4.67 (d, 1H, J ) 7.9 Hz), 4.43 (bq, 1H, J ) 6.6 Hz), 4.22-
4.17 (m, 2H), 4.10 (bs, 1H), 4.08 (d, 1H, J ) 3.1 Hz), 3.93 (dt, 1H, J
) 9.7, 6.8 Hz), 3.91 (d, 1H, J ) 7.9 Hz), 3.88 (dd, 1H, J ) 10.4, 3.3
Hz), 3.85-3.81 (m, 2H), 3.79-3.70 (m, 6H), 3.66 (dt, 1H, J ) 9.7,
6.8 Hz), 2.03 (s, 3H), 1.89 (m, 1H), 1.79 (m, 1H), 1.65-1.54 (m, 3H),
1.38-1.16 (m, 14 H), 0.91-0.85 (m, 6H); 13C NMR (125 MHz, D2O)
δ 175.4, 101.7, 99.0, 92.5, 84.8, 77.9, 74.3, 72.8, 72.0, 71.7, 70.5, 69.3,
69.0 (2 × C), 67.8, 66.8, 62.0, 61.3, 51.1, 34.7, 31.9, 29.8, 29.4, 29.2,
26.3, 23.0, 22.8, 16.2 (2 × C), 14.9, 14.2; HR-ESMS calcd for C31H57-
NO15Na (M + Na+) 706.3626, found 706.3621.
Acknowledgment. This research was supported by grants
from the Natural Sciences and Engineering Research Council
of Canada (to O.H.) and an NSERC/NRC partnership grant (to
M.M.P.) in collaboration with SYNSORB Biotech Inc. X.Q. is
grateful to the Alberta Research Council for a graduate
scholarship in Carbohydrate Chemistry. We gratefully acknowl-
edge Dr. Warren W. Wakarchuk, National Research Council
of Canada, Ottawa, for providing R1,4-galactosyltransferase. We
thank Ms. Catharine A. Compston, Deanne Cole, and Iwa Kong
for their helpful assistance in the isolation of the enzymes, Dr.
A. Morales-Isquierdo for measuring the mass spectra, and Dr.
Hasan Tahir for proofreading of the manuscript.
Supporting Information Available: 1H NMR spectra for
compounds 1-4, 6-12, 14, 15, 19-24 and HMBC spectra for
compounds 19-24 (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
JA993004G