A Unique Chemoenzymatic Synthesis of α-Galactosyl Epitope Derivatives Containing Free Amino Groups: Efficient Separation and Further Manipulation

Article abstract of DOI:10.1021/jo990159y

A novel chemoenzymatic approach for the synthesis of oligosacchrides containing free amino groups has been developed in which thermophilic glycosidases and a fusion enzyme containing catalytic domains of uridine-5′-diphospho-galactose 4-epimerase and α(1→3) galactosyltransferase were used. This methodology, in conjunction with a convenient purification procedure employing ion exchange chromatography, facilitates the lengthy and high-cost process of carbohydrate synthesis. The prepared oligosaccharides range from disaccharide lactosamine (1a), trisaccharide α-Gal-(1→3)-β-Gal-(1→4)-GlcNH₂ (2), tetrasaccharide β-Gal-(1→4)-β-GlcNH ₂-(1→3)-β-Gal-(1→4)-β-Glc-N₃ (7), to pentasaccharide α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNH ₂-(1→3)-β-Gal-(1→4)-β-Glc-N₃ (15). Compounds 2 and 15 are derivatives of natural α-Gal epitopes. Both of them have shown comparable activities with their natural parent compounds toward human anti-Gal IgG. This method provides a practical approach for the preparation and purification of oligosaccharides containing free amino groups, which can be further derivatized for the enhancement of biological activities.

Full text of DOI:10.1021/jo990159y

J . Org. Chem. 1999, 64, 4089-4094
4089
A Un iqu e Ch em oen zym a tic Syn th esis of r-Ga la ctosyl Ep itop e
Der iva tives Con ta in in g F r ee Am in o Gr ou p s: Efficien t Sep a r a tion
a n d F u r th er Ma n ip u la tion
J ianwen Fang, Xi Chen, Wei Zhang, J ianqiang Wang, Peter R. Andreana, and
Peng George Wang*
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
Received J anuary 28, 1999
A novel chemoenzymatic approach for the synthesis of oligosacchrides containing free amino groups
has been developed in which thermophilic glycosidases and a fusion enzyme containing catalytic
domains of uridine-5′-diphospho-galactose 4-epimerase and R(1f3) galactosyltransferase were used.
This methodology, in conjunction with a convenient purification procedure employing ion exchange
chromatography, facilitates the lengthy and high-cost process of carbohydrate synthesis. The
prepared oligosaccharides range from disaccharide lactosamine (1a ), trisaccharide R-Gal-(1f3)-â-
Gal-(1f4)-GlcNH2 (2), tetrasaccharide â-Gal-(1f4)-â-GlcNH2-(1f3)-â-Gal-(1f4)-â-Glc-N3 (7), to
pentasaccharide R-Gal-(1f3)-â-Gal-(1f4)-â-GlcNH2-(1f3)-â-Gal-(1f4)-â-Glc-N3 (15). Compounds
2 and 15 are derivatives of natural R-Gal epitopes. Both of them have shown comparable activities
with their natural parent compounds toward human anti-Gal IgG. This method provides a practical
approach for the preparation and purification of oligosaccharides containing free amino groups,
which can be further derivatized for the enhancement of biological activities.
In tr od u ction
phy (IEC),8 HPLC,7 and charcoal adsorption,8 have been
evaluated for isolation efficiency. SEC, which is conceiv-
ably the most used method for separating enzyme-
synthesized oligosaccharides, is limited to compounds
which have significant molecular weight differences.
Biomolecules such as nucleic acids and proteins are
frequently isolated utilizing IEC. The use of IEC in
carbohydrate chemistry was reported, exploiting the
weakly acidic character of polyhydroxy groups of “neu-
tral” sugars.9 In principle, amino oligosaccharides should
be easy to isolate with IEC since the unprotected amino
group would have a strong affinity to the cation-exchange
resin.8 However, such amino/resin interactions will be
lost if amino-protected monosaccharides or oligosaccha-
rides are used in synthetic procedures as previously
reported in the literature.4
R-Galactosyl epitopes (R-Gal),2 which include trisac-
charides [R-Gal-(1f3)-â-Gal-(1f4)-â-Glc-R] and [R-Gal-
(1f3)-â-Gal-(1f4)-â-GlcNAc-R] and pentasaccharide
[R-Gal-(1f3)-â-Gal-(1f4)-â-Glc-(1f3)-â-Gal-(1f4)-â-
GlcNAc-R] (all structures bearing a R-Gal-(1f3)Galâ
terminus), are abundantly expressed on the cell surface
of most mammals with the exception of humans, apes,
and other Old World primates. On the contrary, anti-
Gal antibodies exist only in these primates. Studies
concluded that the major hyperacute rejection during
xenotransplantation was caused by R-Gal epitopes bind-
ing specifically to human anti-Gal antibodies (anti-Gal).
This discovery has prompted experimental attempts to
overcome hyperacute rejection by either depleting the
recipient’s anti-Gal through R-Gal immobilized affinity
columns or antagonizing anti-Gal by infusing soluble
synthetic R-Gal oligosaccharides.2b,10 Our laboratory has
developed an effective chemoenzymatic method to syn-
Aminosugars, e.g. glucosamine, galactosamine, and
mannosamine, are widely distributed in living organisms
where they constitute building blocks of glycoconjugates
such as glycopeptides and glycolipids.1 They are found
in milk, in blood group substances, and in lipopolysac-
charide antigens, where they serve as part of the cell
surface antigenic determinants (epitopes).2 The chemical
synthesis of oligosaccharides containing aminosugar
moieties (amino oligosaccharides) relies on glycosylation
employing the oxazoline method or phthalimido protect-
ing scheme.3 Recently, enzyme-catalyzed synthesis of
oligosaccharides has evolved into a powerful shortcut for
those chemical strategies.4 Nevertheless, the advantages
of enzymatic synthesis are sometimes overshadowed by
the difficulty of isolating the product(s) from the reaction
mixture normally containing very similar compounds.5
Various separation methodologies, such as size exclusion
chromatography (SEC),4,5,6,7 ion exchange chromatogra-
(1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Ferzi, T.; Solomon, J .
C.; Yuen, C.-T.; J eng, K. C. G.; Frigeri, L. G.; Hsu, D. K.; Liu, F.-T.
Biochemistry 1994, 33, 6342.
(2) (a) Cooper, D. K. C.; Koren, E.; Oriol, R. Immunol. Rev. 1994,
141, 31. (b) Galili, U. Immunol. Today 1993, 14, 480. (c) Sandrin, M.
S.; Vaughan, H. A.; McKenzie, I. F. C. Transplant. Rev. 1994, 8, 134.
(d) Samuelsson, B. E.; Rydberg, L.; Breimer, M. E.; Backer, A.;
Gustavsson, M.; Holgersson, J .; Karlsson, E.; Uyterwaal, A.-C.; Cairns,
T.; Welsh, K. Immunol. Rev. 1994, 141, 151. (e) Nature (Special Issue)
1998, J anuary, 391, 309. (f) Platt, J . L. Nature 1998, 392, 11 (Supp).
(3) Banoub, J .; Boullanger, P.; Lafont, D. Chem. Rev. 1992, 92, 1167.
(4) (a) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T.
Angew. Chem., Int. Ed. Engl. 1995, 34, 412 and 521. (b) Toone, E. J .;
Simon, E. S.; Bednarski, M. D.; Whitesides, G. M. Tetrahedron 1989,
45, 5365. (c) Takayama, S.; Wong, C.-H. Bioorg. Med. Chem. Lett. 1996,
6, 3. (d). Wong, C.-H.; Haynie, S. L.; Whitesides, G. M. J . Org. Chem.
1982, 47, 6.
(5) Kimura, T.; Takayama, S.; Wong, C.-H. Angew. Chem., Int. Engl.
1996, 35, 2348.
(6) Nilsson, K. G.; Eliasson, A.; Lorek, U. Biotechnol. Lett. 1995,
17, 717.
(7) Iwanowitsch, A.; Friebolin, H.; Carganico, R.; Errenst M.;
Weissharr, G.; Brunner, H.; Mann, H. Biol. Chem. 1998, 379, 737.
(8) Samain, E.; Drouillard, S.; Heyraud, A.; Driguez, H.; Geremia,
R. A. Carbohydr. Res. 1997, 302, 35.
(9) Rochlin, R. D.; Pohl, C. A. J . Liq. Chromatogr. 1983, 6, 1577.
10.1021/jo990159y CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/12/1999

4090 J . Org. Chem., Vol. 64, No. 11, 1999
Fang et al.
Sch em e 1
Sch em e 2
eluent, followed by anion-exchange resin to neutralize the
acid with yields of 19% and 23%, respectively. Glu-
cosamine was recovered by eluting with 0.6 M HCl while
the resin was simultaneously regenerated. The reaction
with Gly001-09 was scaled up to a multigram level and
afforded a similar yield. Thermophilic enzyme Gly001-
08 rendered a â(1f6)-linked isomer 1b in 10% yield and
1a as a minor product (3%). It is noteworthy that the
reactions with thermophilic enzymes as catalysts were
conducted at 85 °C or even 90 °C. The conventional
galactosidase from B. circulans also gave the identical
isomers in decreased regioselectivity (1a , 10% yield; 1b,
6% yield) at room temperature. Since these regioisomers
have the same molecular weight, it is almost impossible
to separate them using SEC. Fortunately, individual
isomers were successfully isolated with IEC using a
dilute concentration of HCl (0.05 M).
To further demonstrate the potential of IEC, the
trisaccharide R-Gal-(1f3)-â-Gal-(1f4)-GlcNH3Cl 2 was
prepared from 1a . A fusion enzyme14 (GalE-GalT) con-
taining both uridine-5′-diphospho-galactose 4-epimerase
(GalE) and R(1f3) galactosyltransferase (GalT) was used
as a catalyst with UDP-glucose (UDP-Glc) as a glycosy-
lation donor. This enzyme showed excellent regio- and
stereoselectivity (Scheme 2). The desired product 2 was
isolated in 64% yield using IEC (0.05 M HCl as eluent),
and the starting material was recovered in 22% yield
(0.15 M HCl). GalE-GalT was constructed by in-frame
fusion of the Escherichia coli gene GalE to the 3′-
terminus of a truncated bovine GalT gene within a high-
expression plasmid. It is particularly noteworthy that
this enzyme has two functions: one as an epimerase and
the other as a galactosyltransferase. It uses relatively
inexpensive UDP-glucose (UDP-Glc) instead of high-cost
UDP-Gal as a donor. Three galactosidases, R-galactosi-
dase from Aspergillus niger, R-galactosidase from Green
Coffee Bean, and Gly001-10, were evaluated to catalyze
a transgalactosylation between lactose and 1a . Although
all three galactosidases exhibited catalytic activity after
the reactions were conducted over long periods, the yields
were quite low and a mixture of 2 and its regioisomer
was recovered.
thesize R-Gal epitopes utilizing R(1f3)-galactosyltrans-
ferase.11 More recently we have focused our attention on
chemoenzymatic synthesis of R-Gal analogues containing
versatile handles, such as a free NH2 group,12 which can
be used for further synthetic manipulation to generate
diversity. These derivatives would present the possibility
of identifying unnatural ligands with enhanced binding
affinity toward anti-Gal antibodies. This paper reports
a unique chemoenzymatic preparation of oligosaccharides
with free amino groups ranging from disaccharides to
pentasaccharides. In this synthetic process, saccharides
bearing free amino groups were used as acceptors of
enzyme-catalyzed glycosylations, involving thermophilic
glycosidases and fusion enzymes, to produce more com-
plex glycoconjugates. This novel approach, coupled with
IEC methodology, allows for a convenient way of syn-
thesizing and purifying amino saccharides that could be
used as intermediates for further derivatization.3,13
Resu lts a n d Discu ssion
Disaccharide 1a , lactosamine, which can be found not
only in R-Gal epitopes but also in the core structure of
many glycoproteins and glycolipids, was chosen as the
primary target. â(1f4) Galactosyltransferase-catalyzed
glycosylation, which is convenient and gives definite
regio- and stereoselectivity, was first employed using
UDP-galactose (UDP-Gal) as donor and glucosamine as
acceptor. Unfortunately no significant preferred product
was obtained. A library of thermophilic glycosidases from
Diversa Corporation,12 which included 10 enzymes, as
well as a common galactosidase from Bacillus circulans,
was then screened. The results indicated that two ther-
mophilic enzymes, Gly001-06 and Gly001-09, had excel-
lent regioselectivity toward glucosamine (Scheme 1). Both
enzymes gave the desired product 1a with a â(1f4)-
linkage. Compound 1a was isolated and purified on a
cation-exchange resin column using 0.15 M HCl as
The preparation of tetrasaccharide [â-Gal-(1f4)-â-
GlcNH2-(1f3)-â-Gal-(1f4)-â-Glc-N3 HCl] 7 began with
the glycosylation of 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-â-D-glucopyranosyl bromide 315 and O-(2,4,6-tri-O-
acetyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-acetyl-â-
D-glucopyranosyl azide 416 to afford trisaccharide 5 in a
(10) Rieben, R.; Von Allmen, E.; Korchagina, E.; Nydegger, U.;
Neethling, F. A.; Kujundzic, M.; Koren, E.; Bovin, N.; Cooper, D. K. C.
Xenotransplantation 1995, 2, 98.
(11) Fang, J .; Li, J .; Chen, X.; Zhang, Y.; Wang, J .; Guo, Z.; Zhang,
W.; Yu, L.; Wang, P. G. J . Am. Chem. Soc. 1998, 120, 6636.
(12) Fang, J .; Xie, W.; Li, J .; Wang, P. G. Tetrahetraon Lett. 1998,
329, 919.
(13) Liang, R.; Yan, L.; Loebach, J .; Ge, M.; Uozumi, Y.; Sekanina,
K.; Horan, N.; Gildersleeve, J .; Thonpson, C.; Smith, A.; Biswas, K.;
Still, W. C.; Kahne, D. Science 1996, 274, 1520.
(14) Chen, X.; Fan, H.; Zhang, Y.; Brew, K.; Wang, P. G. Manuscript
in preparation.
(15) Lemieux, R. U.; Takeda, T.; Chung, B. Y. ACS Symp. Ser. 1976,
39, 90.
(16) Zhang, W.; Wang, J .; Li, J .; Yu, L.; Wang, P. G. J . Carbohydr.
Chem. 1999, submitted.

Synthesis of R-Galactosyl Epitope Derivatives
J . Org. Chem., Vol. 64, No. 11, 1999 4091
Sch em e 3
tosylated with GalE-GalT. Compound 15 was isolated
with IEC in an 87% yield (Scheme 5).
To evaluate the consequence of the substitution of
acetamido group in R-Gal epitopes with free amino group,
compounds 2 and 15 along with their parent R-Gal-(1f3)-
â-Gal-(1f4)-GlcNHAc 16 and R-Gal-(1f3)-â-Gal-(1f4)-
â-GlcNHAc-(1f3)-â-Gal-(1f4)-â-Glc-N3 1711 were sub-
jected to an ELISA assay.23 The results demonstrated
compounds 2 and 15 had comparable inhibition activities
toward human anti-Gal IgG with their parent compounds
16 and 17 (Scheme 6). This indicated the free amino
groups did not significantly change the binding ability
of R-Gal epitopes to the antibody. Work is in progress to
attach a variety of functional groups on the amino group
and generate a large number of R-Gal derivatives to
screen for the tight-binding structures.
Con clu sion
Enzyme-catalyzed glycosylation to produce amino sugar
moieties is a powerful tool in the field of carbohydrate
chemistry. This efficient stereo- and regioselective enzy-
matic method is being exploited to prepare oligosaccha-
rides with unprotected amino groups, producing products
ranging from disaccharide to pentasaccharide. This novel
approach, combined with IEC methodology, allows for a
facile way to synthesize and purify amino saccharides.
Upon isolation of these amino oligosaccharides, further
chemical or enzymatic manipulation will lead to more
complex molecules for identification of ligands with
enhanced biological activities.
yield of 73% (Scheme 3). The azido group was purpose-
fully introduced for its synthetic flexibility in the solid-
phase synthesis of glycopeptides,17 glycopolymers, and
glycodendrimers.18 It can also be transformed to other
useful glycosylation functionalities such as glycosyl fluo-
rides for orthogonal oligosaccharide synthesis.19 Depro-
tection of 5 using a 40% methylamine aqueous solution
afforded 6 in 85% yield. Compound 6 did not undergo
glycosylation with UDP-Gal and â(1f4)galactosyltrans-
ferase; however, it still served as a substrate for the
thermophilic enzyme Gly001-09 to yield an important
intermediate amino tetrasaccharide 7 in 11% yield after
purification with IEC.
In comparison, tetrasaccharide 7 was also prepared on
the basis of chemical transformation (Scheme 4). In a
subsequent synthetic scheme, peracetyllactose 8 was
chemically transformed to its azido derivative 9.20 Via
an efficient and simple isopropylidenation using acetone
and trimethylsilyl chloride (TMSCl),21 9 was converted
to the desired 3′,4′-O-isopropylidene derivative 10. Ben-
zoylation of the free hydroxyl groups in 10, followed by
deisopropylidenation in a 60% acetic acid solution, ren-
dered compound 12. Compound 1a was modified to
Schmidt type donor 13.22 Compounds 12 and 13 were
then coupled through a regioselective Schmidt glycosy-
lation to give 14 in 86% yield. Deprotection of 14 with
methylamine afforded 7 in 92% yield.
Exp er im en ta l Section
1
Gen er a l. H and 13C NMR spectra were recorded on a 400
or 500 MHz spectrometer. Mass spectra (FAB or ESI) were
run at the mass spectrometry facility at the University of
California, Riverside. Thin-layer chromatography was con-
ducted on Baker Si250F silica gel TLC plates with a fluorescent
indicator. The library of thermophilic glycosidases was a gift
from Diversa Corporation (San Diego, CA). Bovine serum
albumin (BSA), Type AB human serum, peroxidase conjugated-
goat anti-human IgG antibody, and mouse laminin (a base-
ment membrane glycoprotein containing 50-70 R-Gal epitopes
per molecule) were purchased from Sigma. Other reagents are
all from commercial available sources.
Gen er a l P r oced u r e of Ion Exch a n ge Ch r om a togr a -
p h y. The reaction mixture was loaded onto a cation-exchange
column after the reaction was conducted for a period of time.
Unreacted starting materials and byproducts, which did not
contain amino groups, were removed with distilled water, and
the column was washed with a dilute HCl solution. The
concentration of HCl was dictated by the type of product:
monosaccharide, 0.6 M; disaccharide, 0.15 M; 0.05 M HCl was
required for the isolation of regioisomers 1a and 1b; trisac-
charide, 0.06 M; tetrasaccharide, 0.025 M; pentasaccharide,
0.01 M. The elution was monitored by TLC. The fractions
containing the desired products were collected and neutralized
with anion-exchange resin (Dowex, Marathon WBA, OH- form,
20-50 mesh). After filtration, the anion-exchange resin was
washed with distilled water. The combined solution was
concentrated under reduced pressure and then lyophilized to
give the pure product as a hydrochloride salt.
To prepare pentasaccharide [R-Gal-(1f3)-â-Gal-(1f4)-
â-GlcNH2-(1f3)-â-Gal-(1f4)-â-Glc-N3 HCl] 15, com-
pound 7 from either Scheme 3 or Scheme 4 was galac-
Gen er a l P r oced u r e of Glycosid a se-Ca ta lyzed Glycosi-
d a tion . To a mixture of lactose (20 mmol) and glucosamine
hydrochloride (2 mmol) was added the following quantity of
enzyme [Gly001-06, 2 mL (1.1 mg/mL); Gly001-08, 2 mL (1.9
mg/mL); Gly001-09, 2 mL (1.8 mg/mL); or galactosidase from
(17) Kunz, H.; Dombo, B. Angew. Chem., Int. Ed. Engl. 1988, 27,
711.
(18) Roy, R. Curr. Opin. Struct. Biol. 1996, 6, 692.
(19) Broder, W.; Kunz, H. Bioorg. Med. Chem. 1997, 5, 1.
(20) Tropper, F. D.; Andersson, F. O.; Braun, S.; Roy, R. Synthesis
1992, 618.
(21) Nishida, Y.; Thiem, J . Carbohydr. Res. 1994, 263, 295.
(22) Sadozai, K. K.; Nukada, T.; Ito, Y.; Nakahara, Y.; Ogawa, T.
Carbohydr. Res. 1986, 157, 101.
(23) Galili, U.; Ron, M.; Sharon, R. J . Immunol. Methods 1992, 151,
117.

4092 J . Org. Chem., Vol. 64, No. 11, 1999
Fang et al.
Sch em e 4
D-glu cop yr a n ose (1a ): 1H NMR (D2O, selected data) δ ) 5.31
(d, J ) 3.6 Hz, 1-H R), 4.85 (d, J ) 8.4 Hz, 1-H, â), 4.34 (d, J
) 7.6 Hz, 1′-H, â); 13C NMR (D2O, selected data) δ ) 103.30
(1′-C, â), 92.80 (1-C â), 89.11 (1-C R); HRFABMS calcd for
Sch em e 5
C
12H24NO10 (M+) 342.1400, found 342.1399. O-â-D-Ga la cto-
p y r a n o s y l-(1f6)-2-a m in o -2-d e o x y -D -g lu c o p y r a n o s e
(1b): 1H NMR (D2O, selected data) δ ) 5.30 (d, J ) 2.8
Hz, 1-H R), 4.85 (d, J ) 8.0 Hz, 1-H â), 4.36 (d, J
)
7.2 Hz, 1′-H, â); 13C NMR (D2O, selected data) δ ) 103.64
(1′-C, â), 93.11 (1-C, â), 89.46 (1-C, R).
Gen er a l P r oced u r e of th e F u sion En zym e Ga lE-Ga lT-
Ca ta lyzed Glycosyla tion . GalE-GalT was added to a Tris-
HCl buffer (100 mM, pH ) 7.0) solution of substrate (40 mM),
UDP-glucose (44 mM), MnCl2 (10 mM), and bovine serum
albumin (BSA) (0.1%). After the mixture was agitated under
argon at room temperature for 3 days, the product was
separated in a cation-exchange column as previously described.
It is important to note that the substrate was also recovered.
Sch em e 6
O-r-D-Galactopyr an osyl-(1f3)-O-â-D-galactopyr an osyl-
(1f4)-O-D-2-a m in o-2-d eoxyglu cop yr a n ose H yd r och lo-
r id e (2). Compound 2 (605 mg, 64% yield) was synthesized
via a GalE-GalT-catalyzed reaction with 1a as the substrate
and then was isolated with IEC as previously described. 1a
(22%) was recovered. 2: 1H NMR (D2O, selected data) δ 4.35
(d, J ) 7.5 Hz, 1H), 4.79 (d, J ) 8.1 Hz), 4.96 (d, J ) 3.6 Hz,
1H), 5.27 (d, J ) 3.6 Hz); 13C NMR (D2O, selected data) δ 88.68,
92.35, 95.21, 102.70; HRFABMS calcd for C18H34NO15 (M+)
504.1928, found 504.1955.
O-(3,4,6-Tr i-O-a cet yl-2-p h t h a lim id o-2-d eoxy-â-D-glu -
cop yr a n osyl)-(1f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a -
n osyl)-(1f4)-2,3,6-tr i-O-a cetyl-â-D-glu cop yr a n osyl Azid e
(5). A suspension of 4 (1.1 g, 1.75 mmol), 4A molecular sieves
(2 g), AgOTf (1.37 g, 5.2 mmol), and collidine (0.7 mL, 5.2
mmol) in anhydrous CH2Cl2 (10 mL) was stirred for 2 h at
room temperature and then cooled to -78 °C. A solution of
compound 3 (2.6 g, 5.2 mmol) in CH2Cl2 (10 mL) was added
dropwise to the cooled suspension. The reaction mixture was
gradually warmed to room temperature and stirred for an
additional 12 h. The reaction mixture was filtered through a
pad of Celite, and the solvent was removed under reduced
B. circulans, 10 mg]. The volume of the solution was adjusted
to 10 mL using a phosphate buffer (50 mM, pH 6.0). The
reactions were conducted at 90 °C for Gly001-06, 85 °C for
Gly001-08 and Gly001-09, and at room temperature for
galactosidase from B. circulans. After the mixtures were
stirred for 3 days (Gly001-06 and Gly001-09), 3 h (Gly001-
08), or 2 h (galactosidase from B. circulans), the product(s)
was isolated with IEC as previously described. The ratios of
regioisomers were calculated from 1HNMR of the isomeric
mixtures and the weights of isolated individual pure com-
pounds. O-â-D-Ga la ctop yr a n osyl-(1f4)-2-a m in o-2-d eoxy-

Synthesis of R-Galactosyl Epitope Derivatives
J . Org. Chem., Vol. 64, No. 11, 1999 4093
pressure. Chromatography of the residue over SiO2 with 5:4
ethyl acetate-hexane afforded 5 (1.24 g, 73%): 1H NMR
(CDCl3) δ 7.81-7.72 (m, 4H), 5.74 (dd, J ) 9.0, 11.0 Hz, 1H),
5.34 (m, 2H), 5.19 (t, J ) 10.0 Hz, 1H), 5.06 (t, J ) 9.0 Hz,
1H), 4.82-4.78 (m, 2H), 4.55 (d, J ) 9.0 Hz, 1H), 4.50 (dd, J
) 2.0, 12.0 Hz, 1H), 4.37 (dd, J ) 2.0, 12.0 Hz, 1H), 4.26 (d, J
) 8.0 Hz, 1H), 4.16-4.02 (m, 5H), 3.81-3.73 (m, 3H), 3.69 (t,
J ) 9.0 Hz, 1H), 3.56 (ddd, J ) 2.0, 5.0, 10.0 Hz, 1H), 2.15 (s,
3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 2.02
(s, 3H), 1.96 (s, 3H), 1.83 (s, 3H), 1.78 (s, 3H); 13C NMR (CDCl3)
δ 170.82, 170.58, 170.43, 170.11, 169.69, 169.60, 169.43,
169.38, 168.60, 134.26, 123.70, 109.85, 100.65, 97.61, 87.66,
75.47, 75.06, 74.83, 72.17, 71.84, 71.13, 70.78, 70.60, 70.04,
68.74, 68.65, 61.75, 61.57, 60.76, 54.49, 20.81, 20.72, 20.66,
20.59, 20.44, 20.37; HRFABMS calcd for C44H52NO25Na (M +
Na) 1059.2818, found 1059.2869.
O-(2-Am in o-2-d eoxy -â-D-glu cop yr a n osyl)-(1f3)-O-(â-D-
ga la ctop yr a n osyl)-(1f4)-â-D-glu cop yr a n osyl Azid e Hy-
d r och lor id e (6). A suspension of 5 (920 mg, 0.88 mmol) in
10 mL of a 40% MeNH2 aqueous solution was stirred at room
temperature for 12 h. The mixture was then concentrated
under reduced pressure to give the residue. The residue was
purified with IEC to afford 6 (420 mg, 85%): 1H NMR (D2O) δ
4.81 (d, J ) 8.4 Hz, 1H), 4.77 (d, J ) 8.8 Hz, 1H), 4.50 (d, J )
8.0 Hz, 1H), 4.18 (d, J ) 3.2 Hz, 1H), 4.00-3.66 (m, 12H),
3.55-3.42 (m, 3H), 3.31 (t, J ) 8.4 Hz, 1H), 2.90 (t, J ) 9.2
Hz, 1H); 13C NMR (D2O) δ 103.21, 103.06, 90.59, 82.53, 78.23,
77.33, 76.66, 75.65. 75.00, 74.43, 73.19, 70.76, 70.16, 68.98,
61.60, 61.03, 60.53, 57.01; HRFABMS calcd for C18H33N4O14
(M+) 529.1993, found 529.2002.
O-(3,4-O-Isop r op ylid en e-â-D-ga la ctop yr a n osyl)-(1f4)-
â-D-glu cop yr a n osyl Azid e (10). A mixture of 9 (600 mg, 1.63
mmol), acetone (10 mL), and trimethylsilyl chloride (4 mL) was
stirred at room temperature under N2. After 3 h, the mixture
was suspended with hexane (10 mL) and evaporated under
reduced pressure to give 10 (660 mg, 100%) without further
purification: 1H NMR (CD3OD) δ 4.56 (d, J ) 8.5 Hz, 1H), 4.37
(d, J ) 8.5 Hz, 1H), 4.20 (d, J ) 3.5 Hz, 1H), 4.06 (t, J ) 6.0
Hz, 1H), 3.95-3.75 (m, 4H), 3.61-3.53 (m, 3H), 3.45 (t, J )
7.5 Hz, 1H), 3.31 (s, 1H), 3.32 (t, J ) 7.5 Hz, 1H), 1.48 (s, 3H),
1.33 (s, 3H); 13C NMR (CD3OD) δ 110.43, 103.41, 91.15, 80.15,
79.60, 77.84, 75.70, 74.66, 74.36, 73.84, 73.74, 61,72, 60.92,
27.72, 25.82; HRFABMS calcd for C15H25N3O10Na (M + Na)
430.1438, found 430.1420.
O-(3,4-O-Isop r op ylid en e-2,6-d i-O-ben zoyl-â-D-ga la cto-
p yr a n osyl)-(1f4)-2,3,6-t r i-O-b en zoyl-â-D-glu cop yr a n o-
syl Azid e (11). After a mixture of 10 (650 mg, 1.59 mmol),
pyridine (6 mL), and BzCl (3 mL) was stirred at 0 °C for 12 h,
it was concentrated under reduced pressure. The residue was
purified on silica gel (CH2Cl2) to give 11 (1.25 g, 85%): 1H NMR
(CDCl3) δ 8.14-7.97 (m, 10H), 7.66-7.31 (m, 15H), 5.77 (t, J
) 9.2 Hz, 1H), 5.39 (dd, J ) 8.8, 9.6 Hz, 1H), 5.17 (t, J ) 7.6
Hz, 1H), 4.81 (d, J ) 8.4 Hz, 1H), 4.66 (dd, J ) 1.6, 12.0 Hz,
1H), 4.64 (d, J ) 8.0 Hz, 1H), 4.53 (dd, J ) 4.4, 12.0 Hz, 1H),
4.31-4.23 (m, 3H), 4.12 (dd, J ) 2.0, 5.6 Hz, 1H), 3.98-3.94
(m, 1H), 3.88-3.84 (m, 1H), 3.75 (dd, J ) 7.6, 11.6 Hz, 1H),
1.55 (s, 3H), 1.29 (s, 3H); 13C NMR (CDCl3) δ 167.08, 166.98,
166.63, 166.25, 166.05, 134.63, 134.49, 134.37, 134.27, 131.04,
130.87, 130.74, 130.65, 130.53, 130.36, 129.84, 129.64, 129.57,
129.57, 129.33, 112.04, 101.23, 89.27, 78.17, 76.27, 75.94,
74.68, 74.22, 73.44, 72.49, 72.34, 63.91, 63.42, 28.56, 27.27;
HRFABMS calcd for C50H45N4O3215Na (M + Na) 950.2748,
found 950.2770.
134.67, 134.63, 134.58, 131.02, 130.84, 130.75, 130.53, 129.78,
129.74, 129.66, 129.57, 101.98, 89.11, 76.80, 76.26, 74.72,
73.85, 73.73, 73.66, 72.14, 69.61, 63.45, 62.92; HRFABMS calcd
for C47H41N4O15Na (M + Na) 910.2435, found 910.2462.
O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-(3,6-d i-a cetyl-2-p h th a lim id o-2-d eoxy- â-D-glu cop yr a n o-
syl)-(1f3)-O-(2,6-d i-O-b en zoyl-â-D-ga la ct op yr a n osyl)--
(1f4)-2,3,6-tr i-O-ben zoyl-â-D-glu copyr an osyl Azide Hydr o-
ch lor id e (14). A suspension of compound 13 (40 mg, 0.052
mmol), compound 12 (46 mg, 0.058 mmol), and 4A molecular
sieves (200 mg) in anhydrous CH2Cl2 (3 mL) was stirred for 2
h at room temperature and then cooled to -78 °C. A 0.5 M
solution of BF3‚Et2O in CH2Cl2 (0.2 mL) was added dropwise
to the cooled suspension, and the reaction mixture was
gradually warmed to 0 °C over a period of 3 h and stirred for
an additional 2 h at 0 °C. The reaction was quenched with 2
drops of Et3N, and the mixture was evaporated under reduced
pressure. Chromatography of the residue over SiO2 in 55:1
toluene-EtOH afforded 14 (72 mg, 86%): 1H NMR (CDCl3) δ
8.10-7.88 (m, 5H), 7.64-7.16 (m, 17H), 7.04 (t, J ) 7.5 Hz,
2H), 5.63 (t, J ) 9.0 Hz, 1H), 5.56 (dd, J ) 7.5, 9.5 Hz, 1H),
5.48 (d, J ) 8.5 Hz, 1H), 5.31-5.23 (m, 3H), 5.09 (dd, J ) 7.0,
10.0 Hz, 1H), 4.95 (dd, J ) 3.0, 10.5 Hz, 1H), 4.68 (d, J ) 9.0
Hz, 1H), 4.51 (d, J ) 8.0 Hz, 1H), 4.48 (d, J ) 8.0 Hz, 1H),
4.38 (d, J ) 11.5 Hz, 1H), 4.28 (dd, J ) 4.5, 12.0 Hz, 1H), 4.22
(dd, J ) 4.5, 11.5 Hz, 1H), 4.16 (dd, J ) 8.5, 1.0 Hz, 1H), 4.08
(t, J ) 8.5 Hz, 1H), 4.04-3.99 (m, 3H), 3.82 (t, J ) 6.5 Hz,
1H), 3.76-3.65 (m, 3H), 3.52 (t, J ) 5.5 Hz, 1H), 2.11 (s, 3H),
2.05 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.79 (s,
3H); 13C NMR (CDCl3) δ 171.04, 170.99, 170.76, 170.22, 169.83,
166.72, 166.46, 166.05, 165.82, 164.71, 134.56, 134.14, 133.98,
133.74, 133.44, 130.58, 130.49, 130.33, 130.24, 130.20, 130.11,
129.94, 129.37, 129.17, 129.11, 128.94, 128.86, 123.82, 101.77,
101.12, 98.90, 88.68, 82.08, 77.42, 75.74, 75.54, 73.50, 73.04,
73.00, 71.87, 71.65, 71.56, 71.27, 71.04, 69.76, 68.27, 67.17,
30.40, 21.39, 21.31, 21.22, 21.07; HRFABMS calcd for C79H76
-
N4O32Na (M + Na) 1615.4340, found 1615.4372.
O-(â-D-Ga la ctop yr a n osyl)-(1f4)-O-(2-a m in o-2-d eoxy -
â-D-glu copyr an osyl)-(1f3)-O-(â-D-galactopyr an osyl)-(1f4)-
â-D-glu cop yr a n osyl Azid e Hyd r och lor id e (7). A suspension
of 14 (60 mg, 0.038 mmol) in 3 mL of 40% MeNH2 was stirred
at room temperature for 12 h. The mixture was then concen-
trated to give the residue. The residue was purified with IEC
to afford 7 (25 mg, 92%). The spectra data were identical with
that of the product prepared with a Gly001-09-catalyzed
transglycosylation between lactose and 6. 7: 1H NMR (D2O) δ
5.01 (d, J ) 8.0 Hz, 1H), 4.76 (d, J ) 8.8 Hz, 1H), 4.50 (d, J )
7.6 Hz, 1H), 4.45 (d, J ) 8.0 Hz, 1H) 4.18 (d, J ) 2.8 Hz, 1H),
3.98-3.63 (m, 20 Hz), 3.52 (t, J ) 8.0 Hz, 1H), 3.30 (t, J ) 8.4
Hz, 1H), 3.16 (t, J ) 9.2 Hz, 1H); 13C NMR (D2O) δ 103.70,
103.14, 100.68, 90.59, 82.36, 78.58, 78.22, 77.33, 76.10, 75.68,
75.64, 75.00, 73.22, 73.13, 71.61, 71.29, 70.74, 69.20, 69.04,
61.77, 61.57, 60.56, 60.25, 56.35; HRFABMS calcd for C24H43
-
N4O19 (M+) 691.2522, found 691.2557.
O-(r-D-Ga la ctop yr a n osyl)-(1f3)-O-(â-D-ga la ctop yr a n o-
syl)-(1f4)-O-(2-am in o-2-deoxy -â-D-glu copyr an osyl)-(1f3)-
O-(â-D-galactopyr an osyl)-(1f4)-â-D-glu copyr an osyl Azide
Hyd r och lor id e (15). Compound 15 (30 mg, 0.034 mmol) was
synthesized in 87% yield from 7 (29 mg, 0.039 mmol) in the
manner as previously described. 15: 1H NMR (D2O) δ 4.98 (d,
J ) 3.5 Hz, 1H), 4.87 (d, J ) 8.5 Hz, 1H), 4.60 (d, J ) 8.0 Hz,
1H), 4.37 (d, J ) 8.0 Hz, 1H), 4.35 (d, J ) 8.0 Hz, 1H), 4.04-
4.02 (m, 3H), 3.85-3.48 (m, 25H), 3.15 (t, J ) 8.0 Hz, 1H),
3.02 (dd, J ) 8.5, 10.5 Hz, 1H); 13C NMR (D2O) δ 103.60,
103.11, 100.66, 96.10, 90.57, 82.33, 78.75, 78.17, 77.82, 77.31,
75.76, 75.62, 74.97, 73.19, 71.49, 71.34, 70.71, 70.21, 69.92,
69.77, 69.01, 68.82, 65.45, 61.72, 61.57, 60.51, 60.27, 56.29;
O-(2,6-Di-O-ben zoyl-â-D-ga la ctop yr a n osyl)-(1f4)-2,3,6-
tr i-O-ben zoyl-â-D-glu cop yr a n osyl Azid e (12). After a sus-
pension of 11 (780 mg, 0.84 mmol) in 60% AcOH (20 mL) was
stirred at 95 °C for 5 h, it was concentrated under reduced
pressure. The residue was purified on silica gel (20:1 CH2Cl2-
MeOH) to give 12 (730 mg, 97%): 1H NMR (CDCl3) δ 8.09-
7.93 (m, 10H), 7.63-7.33 (m, 15H), 5.71 (t, J ) 9.2 Hz, 1H),
5.42-5.33 (m, 2H), 4.80 (d, J ) 8.8 Hz, 1H), 4.63 (d, J ) 7.6
Hz, 1H), 4.60-4.55 (m, 2H), 4.17 (t, J ) 9.6 Hz, 1H), 4.05 (dd,
J ) 6.4, 10.8 Hz, 1H), 3.95 (m, 1H), 3.85 (m, 1H), 3.73 (d, 7.2
Hz, 1H), 3. 62 (dd, J ) 6.4, 10.2 Hz, 1H), 3.54 (t, J ) 6.0 Hz,
1H); 13C NMR (CDCl3) δ 167.56, 167.20, 167.06, 166.95, 166.26,
HRFABMS calcd for
853.3016.
C
30H53N4O24 (M+) 853.3050, found
Isola tion of P olyclon a l An ti-Ga l An tibod y fr om Hu -
m a n Ser u m . Polyclonal anti-Gal antibody was isolated from
commercially available human serum with an R-Gal (im-
mobilized trisaccharide[R-Gal-(1f3)-â-Gal-(1f4)-â-GlcNAc])
affinity chromatography column. Type AB human serum (25
mL) was kept in a 56 °C water bath for 30 min to inactivate

4094 J . Org. Chem., Vol. 64, No. 11, 1999
Fang et al.
the complement. The inactivated serum was then passed
through the column to allow for the complexation between the
anti-Gal antibody and the anti-Gal epitope. After an extensive
wash with phosphate buffered saline (PBS, pH 7.4), the bound
anti-Gal antibody remaining on the column was then eluted
with a glycine-HCl buffer (pH 2.8). The pH value of the elute
was adjusted to 7.2 using 0.1 M NaOH, and the resulting
antibody solution was stored as frozen aliquots (about 200 µg/
mL) in PBS.
ELISA In h ibition Assa y. An ELISA assay was conducted
using mouse laminin as the solid-phase antigen. The purified
human polyclonal anti-Gal antibody was first incubated with
varying concentrations of compounds 2, 15, 16, and 17 for 2 h
at 37 °C with gentle shaking. The mixture (50 µL) was then
added to each microtiter plate well (Immulon 4) containing
mouse laminin (10 µg/mL) and incubated for 1.5 h at room
temperature. After removing any unbound anti-Gal antibody
with PBS (pH 7.4), a secondary antibody (1/1000 peroxidase
conjugated-goat anti-human IgG; 50 µL/well) was introduced
and the newly conjugated antibody complex was incubated for
1 h at room temperature. The substrate (TMB (3,3′,5,5′-
tetramethylbenzidine):H2O2, 9:1; BioRad) specific for the per-
oxidase was added at 100 µL/well, and the enzymatic reaction
was quenched by adding 1 N H2SO4 (100 µL/well). A UV
detector (BioRad Microplate Reader, model 3550-UV, at 450
nm) was implemented to measure the varying concentration
levels. PBS, in addition with the secondary antibody, was used
as a background marker, and anti-Gal antibody with secondary
antibody as maximum staining (0% inhibition). The % inhibi-
tion was calculated with eq 1:
(M - S)/(M - B) ) % inhibition
(1)
S refers to the OD450 reading of the sample with different
concentrations of compounds 2, 15, 16, or 17, B the OD450
reading of the background staining, and M the OD405 reading
of the maximum staining. IC50 was calculated from the curve
of inhibition-concentration.
Ack n ow led gm en t. We acknowledge the generous
support from the Herman Frasch Foundation and the
Hercules Incorporation for our research program.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compounds 1a , 1b, 2, 6, 7, and 15. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O990159Y