Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective Method for Kd Determination and Screening of α-Gal Derivatives Binding to Anti-Gal Antibodies (IgG)

Article abstract of DOI:10.1081/CAR-120025323

Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was developed as an effective method for rapid determination of Kd values for α-Gal derivatives binding to human anti-Gal IgG antibodies. Using this method, K_d values fo

Full text of DOI:10.1081/CAR-120025323

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Frontal Affinity Chromatography Coupled to
Mass Spectrometry: An Effective Method for Kd
Determination and Screening of α‐Gal Derivatives
Binding to Anti‐Gal Antibodies (IgG)
Dr. Jianqiang Wang a b c , Boyan Zhang a , Jianwen Fang b , Keiko Sujino a , Hong Li a , Albin
Otter a , Ole Hindsgaul a , Monica M. Palcic a & Peng George Wang b
a Department of Chemistry , University of Alberta , Edmonton, Alberta, T6G 2G2, Canada
b Department of Chemistry , Wayne State University , Detroit, Michigan, 48202, USA
c Triad Therapeutics, Inc. , 9381 Judicial Drive, San Diego, California, 92121, USA
Published online: 23 Aug 2006.
To cite this article: Dr. Jianqiang Wang , Boyan Zhang , Jianwen Fang , Keiko Sujino , Hong Li , Albin Otter , Ole Hindsgaul ,
Monica M. Palcic & Peng George Wang (2003) Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective
Method for Kd Determination and Screening of α‐Gal Derivatives Binding to Anti‐Gal Antibodies (IgG), Journal of Carbohydrate
Chemistry, 22:6, 347-376, DOI: 10.1081/CAR-120025323
To link to this article: http://dx.doi.org/10.1081/CAR-120025323
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 6, pp. 347–376, 2003
Frontal Affinity Chromatography Coupled to Mass
Spectrometry: An Effective Method for Kd
Determination and Screening of a-Gal
Derivatives Binding to Anti-Gal
Antibodies (IgG)
1
2
1
Jianqiang Wang,1,2, Boyan Zhang, Jianwen Fang, Keiko Sujino,
Hong Li,1 Albin Otter,1 Ole Hindsgaul,1 Monica M. Palcic,1
and Peng George Wang2
*
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
2Department of Chemistry, Wayne State University, Detroit, Michigan, USA
ABSTRACT
Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was
developed as an effective method for rapid determination of Kd values for a-Gal
derivatives binding to human anti-Gal IgG antibodies. Using this method, Kd values
for 23 a-Gal compounds were determined for the first time, including an a-Gal
terminated N-linked oligosaccharide which mimics a single N-glycoform present on
the surface of animal cells. A mixture of eight a-Gal derivatives, a model for an a-Gal
compound library, was successfully screened against this anti-Gal IgG using FAC/MS.
The analyte breakthrough sequence, indicated by the ion chromatogram, reflected the
magnitude of the Kd values, confirming its potential application in the screening of
new a-Gal derivatives and mimetics. Ten a-Gal derivatives were designed and
synthesized chemically or enzymatically. Among the compounds analyzed, trivalent
*
Correspondence: Dr. Jianqiang Wang, Triad Therapeutics, Inc., 9381 Judicial Drive, San Diego,
CA 92121, USA; Fax: (858) 457-7893; E-mail: jwang@triadt.com.
347
DOI: 10.1081/CAR-120025323
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

348
Wang et al.
compound 26 demonstrated the strongest binding affinity to anti-Gal IgG with a Kd
value of 3.1 mM. The a-Gal terminated N-linked oligosaccharide 28 had a Kd value of
8.6 mM.
Key Words: a-Gal; Anti-Gal antibody; Kd determination; Frontal affinity
chromatography.
INTRODUCTION
Xenotransplantation,[1–3] the use of animals instead of humans as a source of
organs, tissues and cells, presents a practical solution to the shortage of donated human
organs for transplantation. However, xenotransplantation has been hampered by
xenograft discordance mainly caused by hyperacute rejection (HAR). Recent studies
indicate that HAR can be triggered by the recognition of naturally occurring human
anti-Gal antibodies to carbohydrate epitopes bearing a Gala1–3Galb terminus[4–10]
(termed a-Gal or a-Gal epitopes) on the surface of xenograft cells. As a result, anti-
body-dependent cell-mediated cytotoxicity by human blood monocytes and macro-
phages along with the cascade of complement-mediated lysis of cells can destroy an
organ in minutes to a few hours. Studies seeking to overcome this immunological
barrier are concentrating on strategies that could eliminate or reduce the interaction
between the a-Gal on tissues and the recipient anti-Gal antibodies. These include anti-
Gal antibody immunoadsorption[11–17] and neutralization,[18–23] depletion or inhibition
of complement in the recipient or expression of human complement-inhibiting pro-
teins,[24–29] genetic engineering of an a-Gal ‘knockout’ donor animal or expression of
other carbohydrate structures to mask the a-Gal epitopes,[30–35] and elimination of
human anti-Gal B cells by a-Gal ricin,[36,37] etc. However, none of these strategies have
so far solved the HAR problem found in xenotransplantation. The ultimate solution is
likely to be a combination of some or all of these approaches.
Immunoadsorption and neutralization of anti-Gal antibodies are two strategies for
vitiating the effects of a-Gal oligosaccharides in xenotransplantation. In immunoad-
sorption, a potential recipient is depleted of anti-Gal by passing their blood through an
a-Gal epitope containing column.[11–17] A number of pig-to-primate organ xenotrans-
plantations following anti-Gal immunoadsorption have been reported. This procedure
was shown to extend the survival of pig organs from 1 to 23 days in baboon.[38] More
recently, it was found to be difficult to deplete all of the anti-Gal antibody due to
antibody heterogeneity. McKane and coworkers[39] found that immunoadsorbents
derived from pentasaccharide 3 or trisaccharide 2 (Figure 1) were effective for IgG
removal in all individuals. For IgM, no single immunoadsorbent achieves complete
removal. For example, pentasaccharide 3 immunoadsorbent only removes anti-Gal IgM
in 30% of individuals. Interestingly, the 3-deoxy-trisaccharide (Gala1-3Galb1-4[3deox-
y]GlcNAc) and 6-deoxy-trisaccharide (Gala1-3Galb1-4[6deoxy]GlcNAc) were the best
immunoadsorbents for IgM removal. These two deoxy derivatives were completely
successful in 73% of individuals. The polymorphic nature of the anti-Gal antibody
repertoire poses a considerable challenge when trying to develop an efficient antibody
removal system. So far the best system is the combination of disaccharide 4 and

FAC/MS: Effective Method for Kd Determination
349
Figure 1. The structures of major a-galactosyl epitopes.
trisaccharide 2 attached to agarose which appeares to be efficient in removing anti-Gal
IgG and IgM in all individuals.[39] Simpler, cheaper and more efficient structures would
be desirable.
For anti-Gal antibody neutralization, synthetic a-Gal epitopes are infused into the
recipient, so that the anti-Gal antibody in the recipient will be neutralized by these
epitopes and is no longer free to attack the subsequently transplanted pig organ. Simon,
Cooper and coworkers[18] reported the pharmacokinetic parameters for the intravenous
administration of tri- and pentasaccharides 1 and 3 in the inhibition of hyperacute
rejection of pig hearts transplanted into baboons. The circulatory pharmacokinetics of 1
and 3 were typical of small water-soluble molecules. Their half-lives in serum were
45–50 min, which indicated rapid renal clearance. In the period during which the blood
oligosaccharide concentration was greater than 1 mM (initially maintained at 3–12 mM
for more than 4 h), the serum cytotoxic activity against porcine cells was completely
abolished. This work pointed out several obstacles that need to be overcome before
this approach becomes practical. Firstly, large-scale production of a-Gal oligosaccha-
rides[23,40–50] is needed to support more biological testing. Secondly, the affinity of
anti-Gal antibodies for a-Gal oligosaccharides is not high. Thirdly, the a-Gal oligo-
saccharides are cleared from the blood too rapidly. Fourthly, the heterogeneity of anti-
Gal antibodies leads to inefficient binding. Therefore, finding molecules that bind to
anti-Gal antibody with high affinity is a priority.
Recently, we reported a series of neoglycopolymers with a polyacrylamide backbone
bearing varying densities of a-Gal epitopes.[51] The binding affinity of the a-Gal epitope
for the anti-Gal antibody was dramatically increased by the "multivalent effect". The
IC50 values of some of these a-Gal polymers suggested nM binding to IgM and IgA.
More progress has also been made by others with non-toxic a-Gal polymers such as
a-Gal polylysine conjugate (GAS 914).[52] However, these are impractical because of
their high molecular weights. Small a-Gal molecules, dendrimers[53–56] or well-
defined a-Gal mimetics with high binding affinity may be a better choice.
Current assays for binding to anti-Gal involve traditional one-compound, one-assay
formats. These are slow, thus limiting the number of compounds that can be tested and
exclude the evaluation of compound libraries present as a mixture. Moreover, no
reports are available on the absolute Kd values for interaction of a-Gal derivatives with
anti-Gal subtype IgG, IgM or IgA. Only three compounds have been used to quantify
the interaction of anti-Gal (mixture of IgG, IgA and IgM from a-Gal affinity column)

350
Wang et al.
and a-Gal with Kd values using radioactive equilibrium dialysis.[57] This method is
restricted to a-Gal derivatives. The need for a rapid and quantitative screening method
is clear and we present such a method here.
Frontal affinity chromatography with mass spectrometric detection (FAC/MS) has
been recently developed for the screening of compound mixtures.[58–65] In this method,
a receptor (10 ꢀ 1000 pmol) is immobilized on a suitable support material and packed
into a micro-column (2 ꢀ 40 mL). A mixture containing potential ligands is then
continuously infused through the column. Active ligands will bind to the column until
the capacity of the column becomes saturated, resulting in ligand breaking through at
the infusion concentration. All non-retained compounds will break through earlier in
the void volume of the system. The strongest binding component will appear last in the
chromatogram. The components are identified with high sensitivity (down to 1 pmol/
mL) and analyzed two-dimensionally using an electrospray ionization (ESI) mass
spectrometer. An ion chromatogram is generated from which the breakthrough volume
can be determined. The relationship between the breakthrough volume, V ꢁ V0, and
the concentration of ligand, [x], for the infusion of a single ligand is governed by Eq. 1,
where Bt represents the dynamic binding capacity of the column and Kd is the
dissociation constant of the ligand. This equation indicates that once Bt and [x] are
known, the dissociation constant Kd of the ligand can be determined from a single
measurement of its V ꢁ V0.
V ꢁ V0 ¼ Bt=ð½xŠ þ KdÞ
ð1Þ
V = breakthrough volume of bound ligand (mL)
V0 = breakthrough volume of inactive compound (void marker) (mL)
Bt = binding capacity of the column (pmol)
[x] = concentration of the ligand (mM)
Kd = dissociation constant of the ligand [mM]
Herein, we report the application of FAC/MS to quantify the interaction between a-Gal
derivatives and human anti-Gal IgG antibodies. Using this method, we were able to
determine Kd values of 23 a-Gal derivatives binding to anti-Gal IgG for the first time.
The compounds included chemically and enzymatically synthesized monovalent, di- to
pentasaccharides, dimeric and trimeric ligands as well as a complex 14-mer a-Gal
terminated N-linked oligosaccharides. A small a-Gal mixture was analyzed by this
method to explore the potential application of FAC/MS for screening a-Gal libraries.
RESULTS AND DISCUSSION
Preparation of a Micro-column Containing an
Immobilized Anti-Gal Antibody IgG
The preparation of the anti-Gal IgG FAC/MS column is illustrated in Figure 2.
Human sera (type AB, Sigma) was diluted with PBS (pH 7.4), then passed through an

FAC/MS: Effective Method for Kd Determination
351
Figure 2. Preparation of the anti-Gal IgG FAC/MS column.
affinity column containing synthetic Gala1–3Galb1–4Galb-epitope linked to diatoma-
ceous earth (Synsorb 90, Chembiomed Inc., Edmonton, Alberta, Canada).[66–68] Bound
anti-Gal (IgG + IgM + IgA) were eluted with glycine buffer (pH 2.6) after extensive
washing of the column. The eluate was immediately brought to pH 7 ꢀ 8 with 1 M
Na2CO3. The resulting anti-Gal antibodies were exhaustedly dialyzed against PBS (pH
7.4), then loaded on to a goat anti-human IgG agarose (Sigma) affinity column. After
washing extensively with PBS (pH 7.4), the anti-Gal IgG was eluted with glycine
buffer (100 mM, pH 2.8) and the eluate was immediately neutralized to pH 7 ꢀ 8 with
1 M Na2CO3. The concentration of IgG was estimated by UV absorbance (280 nm).a
Biotinylation of anti-Gal IgG was done by incubating it with NHS-LC-Biotin (Pierce)
in the presence of Gala1–3Galb1–4GlcNAc for 4 hours at room temperature. Gala1–
3Galb1–4GlcNHAc was added to protect the binding site of anti-Gal from bio-
tinylation. The reaction mixture was diluted with PBS buffer (pH 7.4) and then
concentrated by ultrafiltration (NMWL30,000). The process of dilution and ultrafiltra-
tion was repeated 5 times. Alternatively, the biotinylated anti-Gal IgG could be purified
with a goat anti-Gal IgG immunoaffinity column or a-Gal affinity column. The bio-
tinylated IgG was then infused through a pre-packed column containing controlled
porous glass beads with streptavidin immobilized on the surface (CPG-SA) [8.5 cm
long polyetheylene ether ketone (PEEK) column with an internal diameter of 750 mm]
at slow rate (8 mL/min). The flow-through contained unbiotinylated anti-Gal IgG,
which was re-biotinylated and re-infused through the column. The process was repeated
3 times until a preliminary FAC/MS analysis of selected a-Gal derivatives indicated
that sufficient IgG had been immobilized to retard the ligands. Finally, the column was
saturated with a solution of d-biotin in PBS buffer to block unoccupied streptavidin
binding sites.
The column capacity, Bt, was determined by infusing compound 29 through the
column at various concentrations and measuring the corresponding V ꢁ V0 values. A
plot of 1/{[29] (V ꢁ V0)} versus 1/[29] was generated (Figure 3) correlating to
aThe antibodies were purified by the method reported by Galili (see Refs. [66,67]), a method
previously shown to yield only the IgG subtype.

352
Wang et al.
Figure 3. Determination of the column capacity by FAC/MS. Experiment was done with 29 and
data points represent the mean of three replicates.
y = 0.1113x + 0.0272, where the reciprocal of the y-intercept indicates a Bt of 36.8
pmol (1/Bt = 0.0272). The negative reciprocal of the x-intercept indicated a Kd of 4.1
mM for this compound (Kd/Bt = 0.1113). The column was stable even after 300 runs.b
Chemo-enzymatic Syntheses of -Gal Derivatives
A series of a-Gal trisaccharides with differing aglycons were synthesized as shown
in Scheme 1. The synthesis of a-Gal trisaccharides 9a–e started with the trisaccharide
derivative 7 that was prepared from glycosylation of 5 and 6 according to a reported
procedure.[41,51–56] Hydrogenation over PtO2 in methanol, selectively reduced the azido
group of 7 to the primary amine, which was reacted with the corresponding acyl
chlorides to give compounds 8a–d. Compound 8e was obtained by coupling 2-(2-
methoxyethoxy) acetic acid with 7 using 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihy-
droxyquinoline (IIDQ) as a promoter. The fully deprotected a-Gal trisaccharides 9a–e
were obtained from 8a–e after deacetylation and hydrogenation.
The tetrasaccharide 11, Gala1–3Gala1–3Galb1–4GlcNAc–b–(CH2)8COOMe,
was synthesized from 10 enzymatically using bovine a1,3-galactosyltransferase (a1,3-
GalT). As shown in Scheme 2, tetrasaccharide 11 was obtained in 9.6% yield after the
reaction mixture was incubated for 7 days in the presence of an excess of UDP-Gal. The
low yield suggested that acceptor 10, Gala1–3Galb1–4GlcNAcb(CH2)8COOMe, a
product of the same enzyme reaction from Galb1–4GlcNAcb(CH2)8COOMe, is a poor
acceptor for this enzyme. 1H, 2D-GCOSY, 2D-TOCSY and 13C1H–HMQC NMR
studies of 11 show H0001 (5.17 ppm, J = 3.8 Hz), H@1 (5.19 ppm, J = 3.9 Hz), H@3 (4.09
ppm), C@3 (75 ppm). These data support the structure of 11.
Three polyvalent, low molecular weight a-Gal clusters 22, 24, 26 were also
synthesized as illustrated in Schemes 3–5. 1,4-di-{N-[O-a-D-galactopyranosyl-(1 ! 3)-
O-b-D-galactopyranosyl)-(1 ! 8)-3,6-di-oxo-octyl]carbamido}benzene 22 and 1,3,5-tri-
{N-[O-a-D-galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl)-(1 ! 8)-3,6-di-oxo-
bThe column should be stored in a refrigerator (4°C) in PBS (pH 7.4) buffer and warmed to room
temperature before use. The Bt value of the column should be checked and calibrated periodically.

FAC/MS: Effective Method for Kd Determination
353
Scheme 1. Syntheses of trisaccharides 9a–e.
octyl]carbamido}benzene 24 were synthesized chemically starting from galactose
pentaacetate 12. The glycosylation reaction of 12 and 13 was promoted by BF3 ꢂ Et20
˚
in a solution of dichloromethane with 4 A molecular sieves as a water scavenger
(Scheme 3). The reaction gave 14 in 72% yield. Deacetylation of 14 by potassium
carbonate in water and methanol gave 15, which was treated with dibutyltin oxide and
p-methoxybenzyl chloride[69] successively to selectively protect the 3-position of
galactose 15 which was per-O-acetylated. Oxidation with ceric ammonium nitrate
(CAN)[70] removed the methoxybenzyl group to afford acceptor 17.
The coupling reaction of 17 and 18 was promoted by methyl triflate[71] in ether in
˚
the presence of 4 A molecular sieves. The reaction gave the a1,3-linked disaccharide in
Scheme 2. Enzymatic synthesis of tetrasaccharide 11.

354
Wang et al.
Scheme 3. Synthesis of the polyvalent a-Gal derivative 22.
84% yield with an 8/1 (a/b) selectivity, which reacted with NaN3 to form 19. The azido
group of 19 was then selectively reduced by hydrogenation (PtO2 in MeOH) to give the
amine. This amine was immediately used for coupling with terephthaloyl chloride
(Scheme 3) and benzenetricarbonyl chloride (Scheme 4). The coupling reactions
afforded protected dimer 21 (66%) and trimer 23 (62%). Debenzylation and de-
acetylation of 21 and 23 gave the desired products 22 and 24, respectively. The
trivalent a-Gal trisaccharide 26 was obtained in 64% yield from an enzymatic reaction
of the trivalent lactoside 25[72] with UDP-Gal using calf thymus a1, 3-galactosyl-
transferase (Scheme 5).
In order to investigate the interaction of a single complex-N-linked oligosac-
charide[4–10,73–75] commonly found on the animal cell surface with the human anti-
Gal antibody, an a-Gal triantennary N-glycan derivative 28 was also synthesized
(Scheme 6).

FAC/MS: Effective Method for Kd Determination
355
Scheme 4. Synthesis of the polyvalent a-Gal derivative 24.
The tyrosinamide N-glycan 27[76–79] was incubated with an excess of UDP-Gal
and bovine a1,3-GalT[41] for one week at room temperature. The reaction mixture was
filtered through glass wool and then ultrafiltered (NMWL 10,000) to remove the
protein. The filtrate was lyophilized and purified on a Bio-Gel P-4 column. Further
purification was carried out using semi-preparative HPLC. The sample was dissolved in
water and applied to a 1 ꢃ 25 cm semi-preparative C18 reverse phase (RP) HPLC
column, and eluted isocratically at 1 mL/min with 12% acetonitrile. The eluting peaks
detected by UV absorbance at 280 nm were pooled and freeze dried (Figure 4). The
major peak (1) corresponded to the desired a-Gal terminated oligosaccharide 28 with
m/z of 2777 and 1400 representing the M + Na+ and M + 2Na+ peaks, respectively.
Scheme 5. Enzymatic synthesis of the polyvalent a-Gal derivative 26.

356
Wang et al.
Scheme 6. Enzymatic synthesis of a-Gal oligosaccharide 28.
The proton NMR spectrum of 28 possessed readily identifiable Gala, Galb,
GlcNAc and Man anomeric protons as shown in Figure 5. Three anomeric protons
between 5.145–5.151 ppm were assigned to Gala residues G, G’, G@. The resonance
frequencies of the Galb (6’, 8, 6) anomeric protons were significantly influenced by the
attachment of Gala1 residues (G, G’, G@) which resulted in a downfield shift to 4.533–
4.568 ppm from the original 4.458–4.482 ppm in 27.
Determination of Kd Values of -Gal Derivatives by
FAC/MS Analysis and the Screening of an Eight
Compound Mixture Model Library
The dissociation constants (Kd) of a-Gal derivatives binding to anti-Gal IgG were
calculated from Eq. 1 after their corresponding breakthrough volumes, V ꢁ Vo, were
measured by FAC/MS analysis at a fixed concentration ([x]). For operation in the
frontal analysis chromatography mode, the anti-Gal IgG column was washed with
Figure 4. Semipreparative RP–HPLC purification of a-Gal oligosaccharide 28.

FAC/MS: Effective Method for Kd Determination
357
Figure 5. Anomeric proton signals (600 MHz, D2O) for N-linked oligosaccharide 27 (A) and a-
Gal-N-linked oligosaccharide 28 (B). G, G’, G@ (5.145–5.151 ppm); 6’, 8, 6 (4.458–4.482 ppm, A;
4.533–4.568 ppm, B).
ammonium acetate solution (NH4OAc, 2 mM, pH 7.4) to remove the PBS bufferb, and
then directly connected to an electrospray mass spectrometer. The column was flushed
with ammonium acetate buffer continuously until the flow was switched to a second
solution (NH4OAc, 2 mM, pH 7.4) containing a-Gal derivatives (2 mM) and a void
Table 1. Molecular weight and dissociation constants of
synthetic a-gal derivatives.
a-Gal
m/z (MNa+)a
Kd sb [mM]
9a
9b
9c
9d
9e
11
22
24
26
28
630.2
646.3
660.1
8.5 0.7
9.3 1.1
11.6 0.3
13.0 0.6
15.5 0.6
8.9 0.2
8.5 0.2
8.3 0.5
3.1 0.7
8.6 0.7
636.2
642.3
900.5
1099.5
1598.7
1632.7
2777.0
All values are determined from triplicate experiments.
aMonoisotopic molecular weight of the singly charged
sodium adduct.
bDissociation constant with their corresponding standard
deviations.

358
Wang et al.
Table 2. Dissociation constants of other a-Gal derivatives.
m/z
Kd sb
Structure
Compound
(MNa+)a
[mM]
29
917.3
4.1 0.4
8.3 0.4
7.5 0.2
9.8 0.2
6.5 0.5
8.8 0.4
15.7 0.6
19.7 2.5
15.1 2.4
17.6 2.8
12.6 0.5
30
31
32
33
34
35
36
37
38
39
738.4
697.3
651.2
541.2
568.2
608.3
608.3
495.1
495.1
389.1
(continued)

FAC/MS: Effective Method for Kd Determination
Table 2. Continued.
359
m/z
(MNa+)a
Kd sb
Structure
Compound
[mM]
40
41
379.1
379.1
15.1 2.5
8.5 0.4
All values are determined from triplicate experiments with the exception of compound 29 which
was determined from Figure 3.
aMonoisotopic molecular weight of the singly charged sodium adduct.
volume marker (methoxycarbonyloctyl D-Mana1-3-[D-Mana(1–6)]-b-D-mannopyran-
oside, 42, M + Na+, 697.3, 1 mM). The retention volume, (V ꢁ Vvoid
)
marker anti-Gal
,
was obtained by mass spectrometric analysis of the elution front.[58–65]
Compound that binds non-specifically to column elements (walls, beads or protein)
would also demonstrate breakthrough volumes greater than the void volume of the
system, which can potentially introduce errors to Kd values of a given compound. To
control for such errors, a control experiment using the same analyte was carried out in
a parallel blank column. This blank column of CPG-SA had the same size as the anti-
Gal IgG column but contained no immoblized anti-Gal IgG. The control experiment
measured the non-specific retention volume (V ꢁ V
)
void marker blank. Then V ꢁ Vo was
obtained by substracting of the non-specific retention volume (V ꢁ Vvoid marker blank
)
from the retention volume of the ligand in the anti-Gal column (V ꢁ Vvoid marker anti-Gal
)
as shown in Eq. 2.
V ꢁ Vo ¼ ðV ꢁ Vvoid markerÞantiꢁGal ꢁ ðV ꢁ Vvoid markerÞblank
ð2Þ
The dissociation constant (Kd) was generated from Eq. 1 by inputting the observed
V ꢁ Vo volume and chosen [x]. Compound 42 was used as the void marker for all of
the Kd determination experiments except for compound 31 which has the same
M + Na+ value as 42. In this case, n-octyl b-D-glucopyanoside (M + Na+, 315.0) was
selected as the void marker. The Kd values of the synthetic a-Gal derivatives are
summarized in Table 1, while Table 2 shows other a-Gal derivatives.
Comparing the Kd values of all of the monovalent trisaccharides in Tables 1 and 2,
compound 33 showed the strongest binding to anti-Gal IgG with a Kd value of 6.5 mM.
Pentasaccharide 29 (Kd 4.1 mM) has the highest binding affinity of all of the
monovalent a-Gal derivatives analyzed. A modest ‘‘multivalent effect’’ was reflected
by compound 26 with a Kd of 3.1 mM, better than other monovalent trisaccharides. The
a-Gal dimer disaccharide 22 (Kd 8.5 mM) and trimer disaccharide 24 (Kd 8.3 mM)

360
Wang et al.
Figure 6. Frontal chromatogram of the eight compound mixture (A) and the corresponding blank
frontal chromatogram (B).
showed two times higher affinity than the monomer disaccharide 40 (Kd 15.1 mM). The
single a-Gal-N-linked oligosaccharide 28 had a Kd value of 8.6 mM. The Kd values of
these multivalent compounds, i.e. 22, 24, 26, 28 are corrected for valency (i.e. they are
based on the number of galactosyl residues).
To illustrate the potential of FAC/MS screening of a-Gal derivatives and mimetics,
a mixture of eight oligosaccharides, (29, 30, 11, 9c, 9d, 40, 9e and 42, in 2 mM
NH4OAc, pH 7.4), was analyzed. Each of the eight compounds was present at a
concentration of 2 mM, except for compound 42 that was present at 1 mM. The analysis
gave the ion chromatograms shown in Figure 6A, which shows compound 42 breaking
through the column first (left curve), followed by 9e, 40, 9d, 9c, 11, 30 and finally 29.
Compounds 9e and 40 had virtually the same breakthrough volume. The most retarded
compound was the pentasaccharide 29.
To avoid the false indication of activity caused by non-specific binding to column
elements (walls, beads or protein), a control experiment using a blank column was also
carried out. Figure 6B shows the blank column FAC/MS analysis of the eight
compound mixture. It indicates that these eight compounds broke through the column
together with void volume marker 42. Therefore, the breakthrough sequence in Figure
6A is compatible with the magnitude of their Kd values.

FAC/MS: Effective Method for Kd Determination
CONCLUSION
361
In summary, FAC/MS has been shown to be an efficient method to estimate the Kd
values of individual oligosaccharides binding to isolated human anti-a-Gal IgG. The
method is also useful in screening mixtures of oligosaccharides suggesting its ap-
plication in higher-throughput situations such as the screening of small libraries. The
oligosaccharides (di- to pentasaccharides) tested all had Kd values in the range 4.1 – 20
mM, indicating that only modest added interaction takes place beyond the Gal
(a1 ! 3)Gal sequence. Modification of the aglycon in the a-Gal trisaccharide has little
or no effect on binding. Additionally, polyvalency of the ligands had little if any effect
on the bivalent IgG binding. It will be interesting to see if similar behavior will be
observed for IgA and IgM counter parts.
EXPERIMENTAL
General. 1H and 13C spectra were recorded on Varian Mercury 400 NMR,
Varian Unity 500 and Varian Inova 300 and 600 spectrometers. For solutions in D2O,
the residual DOH peak at 300, 400 and 500 MHz was set at 4.67 ppm (ambient
temperature, ca 20–23°C) while at 600 MHz the DOH signal was set at 4.76 ppm at
30°C. FAB high resolution mass spectra were run at the mass spectrometry facility at
University of California, Riverside. ESI high resolution mass spectra were recorded on
a Micro-mass ZabSep Hydroid Sector-TOF at University of Alberta. Baker silica gel
(40mm) was used for column chromatography and E. Merck precoated TLC plates were
used for thin-layer chromatography. Size-exclusion chromatography was performed on
Bio-Gel P2 or P4 supports using distilled water as the eluent. Dialysis was performed
using Spectra/Por molecularporous membrane (16 mm cylinder diameter, molecular
weight cutoff 14 kD). C18 Sep-Pak sample preparation cartridges were from Waters
(Missisauga, ON, Canada). The C18 Reverse phase HPLC column (1 ꢃ 25 cm, dp 5 m)
was from Beckman.
FAC/MS Analysis. All solutions were infused simultaneously using a multi-syringe
pump (PHD 2000, Harvard Apparatus) at a flow rate of 8 mL/min per syringe (1-mL
syringes). A Rheodyne valve (model 9725) was used for flow switching. The column
eluent was combined with the make-up flow (acetonitrile) in a mixing tee,[58–64] to give a
total flow rate of 16 mL/min directed into the Hewlett-Packard series 1100 MSD
electrospray single quadrupole mass spectrometer, operating in the positive-ion mode. A
chamber voltage of ꢁ 4000 V with a grounded electrospray needle, N2 drying gas flow rate
of 4 L/min, and N2 nebulizer pressure of 480 mba were used. Breakthrough volumns were
measured as midpoints in the selected ion chromatograms for each m/z value.
General procedure for the syntheses of trisaccharides 8a–d.[41] Trisaccharide
7 (0.2 mmol) in ethanol (10 mL) was stirred under hydrogen (40 lb/in2) in the presence
of PtO2 (cat.) for 1 h. After the reaction, PtO2 was removed by filtration and the filtrate
was concentrated. To the residue in dry dichloromethane (10 mL) was added TEA (0.6

362
Wang et al.
mmol) and acetyl chloride (0.4 mmol) at –78°C. The mixture was allowed to warm to
room temperature and stirred for 2 h. The solution was washed with 0.2 N HCl,
NaHCO3, brine and dried over Na2SO4. Solvent removal followed by chromatographic
purification on silica gel column afforded 8a–d.
N-[O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-b-D-glucopyranosyl] ben-
zamide (8a). 89%, Rf = 0.20, 2/1 hexane/ethyl acetate, white foamy solid.1H NMR
(400 MHz, CDCl3) d 1.87 (s, 3H, OAc), 1.99 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.09 (s, 3H,
OAc), 2.11 (s, 3H, OAc), 2.14 (s, 3H, OAc), 3.55 (d, J = 6.4 Hz, 2H, H-6a’, H-6a@), 3.74
(t, J = 6.4 Hz, 1H, H-6b’), 3.88 (m, 6H), 4.13 (m, 4H), 4.37 (d, J = 7.6 Hz, 1H), 4.46 (t,
J = 9.6 Hz, 2H), 4.54 (d, J = 11.6 Hz, 2H, PhCH2 ꢁ), 4.75 (m, 3H, H-1, H-1’, H-6a), 4.86
(d, J = 11.6 Hz, 1H, PhCH2 ꢁ), 4.96 (d, J = 11.2 Hz, 1H, PhCH2 ꢁ), 5.02 (t, J = 9.2 Hz,
1H, H-4), 5.11 (brs, 1H, H-1@), 5.17 (dd, J1 = J2 = 8.4 Hz, 1H, H-2), 5.44 (m, 2H, H-2’,
H-3), 5.50 (brs, 1H, H-4’), 7.09 (d, J = 9.2 Hz, 1H, CONH-), 7.30–7.55 (m, 23H, ArH),
7.80 (d, J = 7.6 Hz, 1H, ArH). 13C NMR (100.6 MHz, CDCl3) d 172.9, 171.6, 171.5,
171.4, 170.5, 169.9, 168.2, 139.8, 139.7, 139.1, 133.9, 133.5, 129.9, 129.5(2C), 129.4,
129.3(m), 129.1, 129.0, 128.8 (m), 128.7, 128.6 (m), 128.4, 102.0, 96.1, 79.9, 79.5, 76.7,
76.6, 76.4, 76.0, 75.7, 74.8, 74.4, 74.3, 74.1, 73.3, 72.2, 71.6, 70.9, 69.5, 65.9, 63.3, 62.6,
21.9 (m), 21.6. HR-FABMS Calcd for C65H73NO22Na+ 1242.4522, Found 1242.4511
N-[O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-b-D-glucopyranosyl] acetylsa-
licylamide (8b). 81%, Rf = 0.15, 1:1 hexane/ethyl acetate, white foamy solid.1H
NMR (400 MHz, CDCl3) d 1.86 (s, 3H, OAc), 1.98 (s, 3H, OAc), 2.05 (s, 3H, OAc),
2.06 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.12 (s, 3H, OAc), 2.38 (s, 3H, OAc), 3.54
(d, J = 6.4 Hz, 2H, H-6a@, H-6a’), 3.72 (t, J = 6.4 Hz, 1H, H-6b’), 3.84 (m, 6H), 4.11
(m, 4H), 4.37 (d, J = 8.0 Hz, 1H), 4.45 (m, 2H), 4.53 (d, J = 11.2 Hz, 2H, PhCH2 ꢁ),
4.67 (d, J = 11.6 Hz, 1H, PhCH2 ꢁ), 4.75 (m, 2H, H-1’, H-6a), 4.79 (d, J = 11.6 Hz,
1H, PhCH2 ꢁ), 4.95 (m, 2H, H-1, H-6b), 5.09 (d, J = 3.6 Hz, 1H, H-1@), 5.15 (dd,
J1 = 10.0 Hz, J2 = 18.0 Hz, 1H, H-2’), 5.37 (t, J = 9.0 Hz, 1H, H-2), 5.44 (t, J = 9.0
Hz, 1H, H-3), 5.47 (d, J = 3.2 Hz, 1H, H-4’), 7.08 (d, J = 9.2 Hz, 1H, CONH-), 7.14
(d, J = 8.0 Hz, 1H, ArH), 7.27–7.42 (m, 20H, ArH), 7.52 (t, J = 8.0 Hz, 1H, ArH),
7.77 (d, J = 8.0 Hz, 1H).13C NMR (100.6 MHz, CDCl3) d 172.4, 171.6, 171.5, 171.4,
170.5, 170.1, 169.9, 166.5, 149.6, 139.8, 139.7, 139.1, 133.9, 130.8, 129.5 (m), 129.4,
129.3 (m), 129.1, 128.9 (m), 128.8, 128.7 (m), 128.6, 127.6, 127.4, 124.6, 101.9, 96.1,
79.5, 79.3, 76.8, 76.7, 76.4, 75.9, 75.8, 74.6, 74.4, 74.3, 74.0, 73.6, 72.2, 72.1, 71.6,
70.9, 69.6, 65.9, 63.3, 62.6, 22.2, 21.9 (m), 21.8 (m), 21.6. HR-FABMS Calcd for
C67H75NO24Na+ 1300.4576, Found 1300.4535.
N-[O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-b-D-glucopyranosyl]–o-ani-
soylamide (8c). 90%, Rf = 0.20, 1:1 hexane/ethyl acetate, white foamy solid. 1H
NMR (500 MHz, CDCl3) d 1.84 (s, 3H, OAc), 1.96 (s, 3H, OAc), 2.00 (s, 3H, OAc),
2.05 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.11 (s, 3H, OAc), 3.52 (d, J = 6.5 Hz, 2H,
H-6a@, H-6a’), 3.69 (t, J = 9.0 Hz, 1H, H-6b’), 3.83 (m, 6H), 3.95 (s, 3H, OCH3), 4.00
(dd, J1 = 9.0 Hz, J2 = 3.5 Hz, 1H, H-3@), 4.07 (t, J = 6.0 Hz, 2H), 4.19 (m, 1H), 4.34
(d, J = 8.0 Hz, 1H), 4.41 (d, J = 11.5 Hz, 2H, PhCH2 ꢁ), 4.51 (d, J = 11.5 Hz, 2H,

FAC/MS: Effective Method for Kd Determination
363
PhCH2 ꢁ), 4.66 (d, J = 11.5 Hz, 1H, PhCH2 ꢁ), 4.72 (m, 2H), 4.83 (d, J = 12.0 Hz,
1H), 4.92 (d, J = 11.0 Hz, 1H, 6a), 5.08–5.13 (m, 3H, H-2, H-1@, H-1), 5.34 (m, 1H,
H-2’), 5.48 (m, 2H, H-3, H-4’), 6.95 (d, J = 8.5 Hz, 1H, CONH-), 7.06 (t, J = 7.5 Hz,
1H, ArH), 7.27–7.39 (m, 15H, ArH), 7.48 (dd, J1 = 7.5 Hz, J2 = 9.0 Hz, 1H, ArH),
8.20 (dd, J1 = 2.0 Hz, J2 = 7.5 Hz, 1H, ArH), 8.61 (d, J = 9.0 Hz, 1H, ArH). 13C NMR
(125.7 MHz, CDCl3) d 171.3, 171.1, 170.9, 170.0, 169.4, 166.3, 158.6, 139.4, 138.8,
134.4, 133.3, 129.1, 128.9 (m), 128.8 (m), 128.6, 128.4, 128.3 (m), 128.2, 128.1, 121.9,
120.9, 112.1, 101.6, 95.8, 79.2, 79.0, 76.5, 76.4, 76.1, 75.5, 75.2, 74.3, 74.0, 73.9, 73.8,
73.5, 71.9, 71.3, 71.2, 70.6, 69.2, 65.7, 63.1, 62.2, 56.4, 21.5, 21.4 (m), 21.3, 21.1. HR-
FABMS Calcd for C66H75NO23Na+ 1272.4627, Found 1272.4678.
N-[O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-b-D-glucopyranosyl] cyclo-
hexylamide (8d). 89% (diaisomer 2:1), Rf = 0.25, 2:1 hexane/ethyl acetate, white
1
foamy solid. H NMR (500 MHz, CDCl3) d 1.25 (m, 6H, c-Hex), 1.75 (m, 4H, c-Hex),
1.81 (s, 3H, OAc), 1.91 (s, 3H, OAc), 2.00 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.06 (s, 3H,
OAc), 2.07 (s, 3H, OAc), 2.28 and 2.42 (t, J = 11.0 Hz, 1H, diaisomer 2:1, c-Hex), 3.21
and 3.09 (m, 1H, diaisomer 2:1), 3.48 (d, J = 6.5 Hz, 2H), 3.61–3.81 (m, 7H), 4.03 (m,
3H), 4.29 (d, J = 7.5 Hz, 1H), 4.37 (m, 2H), 4.48 (d, J = 11.0 Hz, 2H), 4.63 (d, J = 11.5
Hz, 1H, PhCH2 ꢁ), 4.69 (m, 2H, PhCH2 ꢁ), 4.80 (m, 2H, PhCH2, H-4), 4.89 (d, J = 11.5
Hz, 1H, H-6a), 5.04 (d, J = 3.0 Hz, 1HH-1@), 5.08 (t, J = 8.0 Hz, 1H, H-2), 5.20 (t,
J = 9.5 Hz, 1H, H-2’), 5.27 (m, 1H, H-3), 5.43 (m, 1H, H-4’), 6.26 (d, J = 9.5 Hz, 1H,
CONH-), 7.24–7.35 (m, 20H, ArH). 13C NMR (125.7 MHz, CDCl3) d 181.1, 176.9 &
175.6 (diaisomer), 172.0, 171.1, 170.9, 170.1, 169.5, 139.2, 138.7, 129.0 (m), 128.8 (m),
128.7, 128.5, 128.4 (m), 128.2, 128.1, 101.5, 95.6, 79.1, 78.6, 76.3, 76.1, 75.9, 75.5,
75.2, 74.3, 73.9(2C), 73.6, 73.0, 71.7, 71.1, 70.5, 69.1, 67.7, 67.6, 67.4, 65.5, 62.9, 62.2,
50.8, 48.3, 46.6 and 45.8, 43.5 and 42.7, 40.9, 37.2, 30.4, 30.2, 29.9, 29.5, 29.4, 26.5,
26.4, 26.3, 26.1. HR-FABMS Calcd for C65H79NO22Na+ 1248.4991, Found 1248.5012.
N-[O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-ace-
tyl-b-D-galactopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-b-D-glucopyranosyl]–2-(2-
methoxyethoxy) acetamide (8e). Trisaccharide 7 (250 mg, 0.21 mmol) in ethanol (10
mL) was stirred under hydrogen (40 lb/in2) for 1 h. After the reaction, PtO2 was
removed by filtration and the filtrate was evaporated. To the residue in dry
dichloromethane (10 mL) was added 2-(2-methoxyethoxy) acetic acid (72 uL, 0.63
mmol) and 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquonoline (IIDQ, 0.38 ml, 1.26
mmol). The mixture was stirred overnight at room temperature. Solvent removal
followed by chromatographic purification on silica gel column (eluent: 1/2 hexane/ethyl
acetate) afforded 8e (0.15 g, 54 %, Rf = 0.45, 1:3 hexane/ethyl acetate) as a white
1
foamy solid. H NMR (500 MHz, CDCl3) d 1.82 (s, 3H, OAc), 1.93 (s, 3H, OAc), 2.01
(s, 6H, OAc), 2.07 (s, 3H, OAc), 2.08 (s, 3H, OAc), 3.40 (s, 3H, OMe), 3.51 (m, 2H,
H-6a@, H-6a’), 3.54 (dd, J1 = 6.5 Hz, J2 = 8.5 Hz, 2H), 3.61 (m, 2H), 3.66 (t, J = 7.0
Hz, 1H), 3.74–3.84 (m, 6H), 3.95–4.12 (m, 6H), 4.31 (d, J = 8.0 Hz, 1H), 4.39 (m,
2H), 4.49 (d, J = 12.0 Hz, 1H, PhCH2 ꢁ), 4.64 (d, J = 11.5 Hz, 1H, PhCH2 ꢁ), 4.69 (m,
2H, PhCH2 ꢁ), 4.81 (d, J = 12.0 Hz, 1H, H-6a), 4.91 (m, 2H, H-1’, H-1), 5.04 (d,
J = 3.0 Hz, 1H, H-1@), 5.09 (dd, J1 = 8.0 Hz, J2 = 10.0 Hz, 1H, H-2), 5.24 (m, 2H, H-
2’, H-3), 5.43 (d, J = 3.0 Hz, 1H, H-4’), 7.24–7.37 (m, 20H, ArH). 13C NMR (125.7
MHz, CDCl3) d171.6, 171.2 (2C), 171.6, 170.9, 170.2, 169.5, 139.4, 139.3, 138.7,

364
Wang et al.
129.1 (m), 129.0 (m), 128.9 (m), 128.8 (m), 128.7, 128.4 (m), 128.3, 128.2, 128.1,
79.1, 76.4, 76.1, 75.9, 75.5, 75.3, 74.3, 74.0, 73.9, 73.6, 73.5, 72.2, 71.7, 71.5, 71.1,
71.0, 70.5, 69.2, 65.5, 62.9, 62.1, 59.7, 21.5, 21.4 92C), 21.3 (2C), 21.1. HR-FABMS
Calcd for C65H77NO24Na+ 1254.4733, Found 1254.4786.
General procedure for the synthesis of trisaccharides 9a–e. Pd/C (10%, 100
mg) was added to a solution of 8a–e (500 mg) in methanol (30 mL). The black
suspension was charged with compressed hydrogen (50 lb/in2) after degassing. The
reaction took 5 h until no more hydrogen was consumed. The mixture was filtered and
the filtrate was evaporated to give a residue that was dissolved in anhydrous methanol
(500 mL) followed by the addition of NaOMe to adjust the pH value of the solution to
9.0. The mixture was stirred for 2 h. Dowex (H+ form) was added to neutralize the
solution. The resin was filtered and the filtrate was concentrated to afford 9a–e as a
white solid after lyophilization. Further purification on Bio-Gel P2 used water as eluent.
N-[O-a-D-Galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 4)-1-b-D-glu-
copyranosyl] benzamide (9a). 97%, Rf = 0.55, 8/2/2 2-methylpropanol/H2O/ethyl
acetate. 1H NMR (500 MHz, D2O) d 3.49 (t, J = 8.5 Hz, 1H, H-2), 3.54–3.88 (m, 15H),
4.06 (m, 2H, H–5@ & H-4’), 4.41 (d, J = 8.0 Hz, 1H, H-1’), 5.01 (d, J = 4.0 Hz, 1H, H-1@),
5.07 (d, J = 9.0 Hz, 1H, H-1), 7.40 (t, J = 7.5 Hz, 2H, ArH), 7.51 (t, J = 7.5 Hz, 1H, ArH),
7.69 (d, J = 8.5 Hz, 2H, ArH). 13C NMR (125.7 MHz, D2O) d 172.3, 133.4, 129.5, 128.1,
103.4, 96.1, 81.4, 78.6, 77.8, 77.1, 75.9, 75.7, 75.2, 72.0, 71.5, 70.2, 69.9, 69.7, 68.8, 65.5,
61.7, 61.6, 60.6. HR-FABMS Calcd for C25H37NO16Na+ 630.2010, Found 630.1987.
N-[O-a-D-Galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 4)-1-b-D-glu-
copyranosyl] salicylamide (9b). 91%, Rf = 0.60, 10/2/2 2-methylpropanol/H2O/ethyl
acetate. 1H NMR (400 MHz, D2O) d 3.53 (t, J = 9.2 Hz, 1H, H-2), 3.60–3.95
(m, 15H), 4.06 (m, 2H, H-4@ & H-3@), 4.11 (m, 2H, H-5@ & H-4’), 4.47 (d, J = 7.6 Hz,
1H, H-1’), 5.07 (d, J = 3.6 Hz, 1H, H-1@), 5.16 (d, J = 9.2 Hz, 1H, H-1), 6.83 (m, 2H,
ArH), 7.36 (m, 1H, ArH), 7.75 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H, ArH).13C NMR
(100.6 MHz, D2O) d 171.4, 162.6, 134.8, 129.4, 119.4, 117.8, 116.6, 102.9, 95.5, 79.5,
78.2, 77.2, 76.4, 75.2, 75.1, 71.8, 70.9, 69.6, 69.3, 69.2, 68.3, 64.9, 61.1, 60.9, 60.1.
HR-FABMS Calcd for C25H37NO17Na+ 646.1959, Found 646.1934.
N-[O-a-D-Galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 4)-1-b-D-glu-
copyranosyl]-o-anisoylamide (9c). 91%, Rf = 0.50, 8/2/2 2-methylpropanol/H2O/
ethyl acetate. 1H NMR (400 MHz, D2O) d 3.57 (t, J = 9.2 Hz, 1H, H-2), 3.65–
4.01 (m, 18H), 4.18 (m, 2H, H-5@ & H-4’), 4.52 (d, J = 7.6 Hz, 1H, H-1’), 5.12 (d,
J = 4.0 Hz, 1H, H-1@), 5.19 (d, J = 9.2 Hz, 1H, H-1), 7.11 (m, 2H, ArH), 7.56 (m, 1H,
ArH), 7.75 (dd, J1 = 1.6 Hz, J2 = 7.6 Hz, 1H, ArH).13C NMR (100.6 MHz, D2O) d
170.1, 157.6, 134.3, 130.5, 121.2, 120.9, 112.5, 103.0, 95.6, 79.7, 78.4, 77.4, 76.6, 75.4,
75.2, 71.8, 71.0, 69.7, 69.4, 69.3, 68.4, 65.0, 61.2, 61.1, 60.2, 56.1. HR-FABMS Calcd
for C26H39NO17Na+ 660.2116, Found 660.2101.
N-[O-a-D-Galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 4)-1-b-D-glu-
copyranosyl] cyclohexylamide (9d). 96%, Rf = 0.70, 8/2/2 2-methylpropanol/H2O/
ethyl acetate. 1H NMR (500 MHz, D2O) d 1.06–1.25 (m, 6H, c-Hex), 1.52–1.69
(m, 4H, c-Hex), 2.16 & 2.55 (t, J = 11.5 Hz, diaisomer, 2/1, 1H, c-Hex), 3.31 (t,

FAC/MS: Effective Method for Kd Determination
365
J = 9.0 Hz, 1H, H-2), 3.45 (t, J = 5.5 Hz, 1H), 3.54–3.82 (m, 13H), 3.88 (d,
J = 3.0 Hz, 1H, H-4@), 4.05 (m, 2H, H-5@ & H-4’), 4.38 (d, J = 7.5 Hz, 1H, H-1’),
4.84 (d, J = 9.0 Hz, 1H, H-1), 5.00 (d, J = 4.0 Hz, 1H, H-1@).13C NMR (125.7
MHz, D2O) d 182.2 & 178.6, 103.4, 96.1, 79.7, 78.7, 77.8, 77.0, 75.9, 75.7, 72.1,
71.5, 70.2, 69.9, 69.8, 68.8, 67.2, 67.1, 65.5, 61.7, 61.6, 60.6, 46.5, 45.6, 42.9, 40.5,
29.6 (m), 29.5, 28.8 (m). HR-FABMS Calcd for C25H43NO16Na+ 636.2479, Found
636.2477.
N-[O-a-D-Galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl-(1 ! 4)-1-b-D-glu-
copyranosyl]–2-(2-methoxyethoxy)acetamide (9e). 81%, Rf = 0.20, 8/2/2 2-methyl-
propanol/H2O/ethyl acetate. 1H NMR (400 MHz, D2O) d 3.34 (s, 3H, OMe), 3.47
(t, J = 9.2 Hz, 1H, H-2), 3.59–3.91 (m, 19H), 3.96 (d, J = 3.2 Hz, 1H, H-4@), 4.12
(m, 3H, H-5@, H-4’, H-3@), 4.47 (d, J = 7.6 Hz, 1H, H-1’), 5.00 (d, J = 9.2 Hz,
1H, H-1), 5.08 (d, J = 3.6 Hz, 1H, H-1@).13C NMR (100.6 MHz, D2O) d 174.0,
102.8, 95.5, 78.9, 77.9, 77.2, 76.5, 75.5, 75.1, 71.4, 70.9, 70.8, 70.2, 69.6, 69.5, 69.3,
69.1, 68.3, 64.9, 61.1, 60.9, 59.9, 58.1. HR-FABMS Calcd for C23H41NO18Na+
642.2221, Found 642.2240.
8-Methoxycarbonyloctyl
-D-galactopyranosyl-(1 ! 3)-a-D-galactopyranosyl-
(1 ! 3)-b-D-galactopyranosyl-(1 ! 4)-b-D-2-deoxy-2-acetamidoglucopyranoside
(11). Trisaccharide 10 (6 mg, 8.4 mmol), UDP-Gal (76.8 mg, 15 ꢃ 8.4 mmol) and 10
mL of alkaline phosphatase (1U/mL) were added to a 2.5-mL solution of bovine a1,3-
Gal T (2.5 U in PBS), MnCl2 (10 mM) and BSA (0.1%). The mixture was incubated
with agitation at room temperature for one week. The reaction mixture was then
filtered through glass wool and loaded onto two sequential C18 Sep-Pak cartridges
which were prewashed successively with 20 mL HPLC MeOH and 20 mL H2O. The
cartridges were washed with water (80 mL) and eluted with MeOH (15 mL). The eluate
was concentrated, redissolved in water, filtered through a Millex-GV 0.22-mM filter,
and lyophilized to yield crude product (5.3 mg) as a white solid. The crude product was
dissolved in a small amount of CH2Cl2/MeOH/H2O (60/40/1) and loaded onto an
IATROBEAD (IATRON Laboratories INC.) column. The product was purified by
chromatography (eluent: 60/40/1 CH2Cl2/MeOH/H2O) to give tetrasacharide 11 (0.7
mg, 9.6%, Rf = 0.45, 65/35/8 CH2Cl2/MeOH/H2O) as a white solid. Unreacted starting
1
material (4.0 mg) was recovered after chromatography. H NMR (600 MHz, D2O) d
1.30 (br., 8H), 1.54 (t, J = 6.0 Hz, 2H), 1.60 (t, J = 7.2 Hz, 2H), 2.03 (s, 3H), 2.39 (t,
J = 7.8 Hz, 2H), 3.57–4.03 (m, 24H), 4.09 (dd, J1 = 3.0 Hz, J2 = 10.2 Hz, H-3@), 4.20
(m, H-4’, H-5@, H-5000)J = 2.4 Hz, H-4@), 4.52 (d, J = 7.8 Hz, H-1), 4.55 (d, J = 7.8 Hz,
H-1’), 5.17 (d, J = 3.6 Hz, H-1000), 5.19 (d, J = 3.6 Hz, H-1@).1H-13C HMQC (600 MHz,
000
D2O) C3@ (75.0 ppm), C4@ (66.3 ppm), C4 (70.0 ppm). HR-ESMS Calcd for
C36H63NO23Na+ 900.3688, Found 900.3693.
1-Chloro-3,6-dioxa-8-octyl 2,3,4,6-tetra-O-acetyl- -D-galactoside (14). To dry
dichloromethane (300 mL) in a 3-neck flask was added galactose pentaacetate 12 (48.0
g, 123 mmol), 2-[2-(2-chloroethoxy)ethoxy]ethanol (13) (31.2 g, 28 mL, 185 mmol)
˚
and 4 A molecular sieves (10 g). After the reaction mixture was stirred for 1 h,
BF3 ꢂ Et2O (52.0 g, 45 mL, 370 mmol) was added dropwise during 3 h and the
resulting mixture was stirred for 48 h. The suspension was filtered through a pad of
celite and the filtrate was poured into a pre-cooled NaHCO3 solution. The organic layer

366
Wang et al.
was separated, and the aqueous solution was extracted twice with dichloromethane. The
combined organic phases were washed with brine, dried over anhydrous Na2SO4 and
concentrated in vacuo to give an oil. The crude product was purified by flash column
chromatography eluting with a mixture of hexane and ethyl acetate (1/1), to give the
product 14 (44.2 g, 72%) as a colorless oil.1H NMR (500 MHz, CDCl3) d 1.93 (s, 3H,
OAc), 1.99 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.10 (s, 3H, OAc), 3.57–3.62 (m, 8H),
3.70 (m, 3H), 3.87–3.91 (m, 2H), 4.06–4.13 (m, 2H), 4.53 (d, J = 8.0 Hz, 1H, H-1),
4.96 (dd, J1 = 3.6 Hz, J2 = 10.4 Hz, 1H, H-3), 5.14 (dd, J1 = 8.4 Hz, J2 = 10.4 Hz, 1H,
H-2), 5.33 (d, J = 3.6 Hz, 1H, H-4). 13C NMR (125.7 MHz, CDCl3 ) d 171.0, 170.9,
170.7, 170.1, 102.0, 72.0, 71.6, 71.3 (m), 71.0, 69.7, 69.5, 67.8, 62.0, 43.4, 21.4, 21.3
(2C), 21.2. HR-FABMS Calcd for C20H31O12ClNa+ 521.1402, Found 521.1426.
1-Chloro-3,6-dioxa-8-octyl -D-galactoside (15). To a 1000 mL flask containing
14 (44.0 g, 88.3 mmol) was added a mixture of methanol (150 mL) and 1 M Na2CO3
aqueous solution (200 mL). The mixture was stirred at room temperature for 36 h. The
reaction solution was poured into a 1000 mL Erlenmeyer flask cooled in an ice–water
bath. Methanol (600 mL) was added and a white solid precipitated. The suspension was
filtered and washed with methanol. The filtrate was neutralized with Dowex resin (H+
form) until pH 6 was achieved. The resin was removed by filtration and washed with
water and the filtrate was concentrated in vacuo. Toluene was added to further remove
water via azeotropic distillation to give 1-chloro-3,6-dioxa-8-octyl b-D-galactoside (15)
1
(26.8 g, 92%). H NMR (500 MHz, D2O) d 3.35 (m, 1H, H-3), 3.47–3.70 (m, 15H),
3.76 (d, J = 3.6 Hz, 1H, H-4), 3.92 (m, 1H, H-5), 4.26 (d, J = 8.0 Hz, 1H, H-1). 13C
NMR (125.7 MHz, D2O) d 103.4, 75.8, 73.3, 71.4, 71.3, 70.3, 70.1, 70.0, 69.2 (m),
61.6, 43.7.
1-Chloro-3,6-dioxa-8-octyl 3-O-p-methoxybenzyl- -D-galactoside (16). Com-
pound 15 (26.6 g, 80.5 mmol) was dissolved in anhydrous methanol (150 mL) to
which dibutyltin oxide (30.1 g, 120.8 mmol) was added. After the suspension was
refluxed for 6 h under an atmosphere of nitrogen, the solution was cooled to room
temperature followed by removal of methanol in vacuo. Then dry benzene (500 mL), p-
˚
methoxybenzyl chloride (35.6 mL, 161.0 mmol), TBAB (13.3 g, 40.2 mmol) and 4 A
MS (10 g) were added. The mixture was refluxed for 2 h under N2, then cooled to 0°C
when diethylamine (20 mL) was added to quench the reaction. The reaction mixture
was diluted with methanol (300 mL), filtered through a pad of celite and concentrated.
The residue was purified by column chromatography (silica gel, eluent: ethyl acetate
then ethyl acetate/ethanol: 10/1, 5/1) to afford 16 (16.7 g, 46%) as colorless oil. 1H NMR
(500 MHz, CDCl3) d 3.35 (dd, J1 = 9.0 Hz, J2 = 3.0 Hz, 1H, H-3), 3.41 (t, J = 5.5 Hz, 1H,
H-5), 3.59–3.86 (m, 17H), 3.93 (d, J = 5.5 Hz, 1H, H-6a), 3.99 (m, 1H, H-4), 4.25 (d,
J = 8.0 Hz, 1H, H-1), 4.63 (s, 2H, PhCH2O ꢁ ), 6.84 (d, J = 9.6 Hz, 2H, ArH), 7.28 (d,
J = 9.6 Hz, 2H, ArH). 13C NMR (125.7 MHz, CDCl3) d 160.0, 130.7, 130.3 (2C), 114.6
(2C), 104.3, 80.6, 75.2, 72.4, 72.0, 71.4, 71.1, 71.0 (m), 69.3, 67.6, 62.6, 56.0, 43.3. HR-
FABMS Calcd for C20H31O9ClNa+ 473.1554, Found 473.1550.
1-Chloro-3,6-dioxa-8-octyl 2,4,6-tri-O-acetyl- -D-galactoside (17). To com-
pound 16 (14 g, 31 mmol) in a 500 mL flask was added a mixture of Ac2O (100
mL) and pyridine (150 mL), followed by addition of DMAP (120 mg). The reaction
was complete after 3 h. The solvents were removed in vacuo to give an oil, which

FAC/MS: Effective Method for Kd Determination
367
was dissolved in chloroform (300 mL). The solution was washed sequentially with 0.5
N HCl, water, NaHCO3 and brine. It was then dried over anhydrous Na2SO4 and
evaporation of the solvent afforded an oil which was purified by column
chromatography (silica gel, hexane/ethyl acetate: 1/3, 1/6, sequentially) to give 1-
chloro-3,6-dioxa-8-octyl 2,4,6-tri-O-acetyl-3-O-p-methoxybenzyl-b-D-galactoside (16.1
1
g, 90%) as an colorless oil. H NMR (500 MHz, CDCl3) d 1.95 (s, 3H, OAc), 1.99
(s, 3H, OAc), 2.06 (s, 3H, OAc), 3.46 (dd, J1 = 9.6 Hz, J2 = 3.6 Hz, 2H, H-3), 3.53–
3.66 (m, 12H), 3.71 (s, 3H), 3.86 (m, 1H, H-5), 4.08 (m, 2H, H-6a,b), 4.24 (d,
J = 11.6 Hz, 1H, PhCH2 ꢁ), 4.38 (d, J = 8.4 Hz, 1H, H-1), 4.54 (d, J = 11.6 Hz, 1H,
PhCH2 ꢁ), 5.00 (dd, J1 = 9.6 Hz, J2 = 8.0 Hz, 1H, H-2), 5.41 (d, J = 2.8 Hz, 1H, H-
4), 6.78 (d, J = 8.6 Hz, 2H, ArH), 7.10 (d, J = 8.6 Hz, 2H, ArH). 13C NMR (125.7
MHz, CDCl3 ) d 171.0, 170.9, 170.0, 160.0, 130.2, 130.1 (2C), 114.4 (2C), 101.9,
76.8, 71.9, 71.6, 71.2 (m), 71.1, 71.0, 69.4, 66.7, 62.6, 55.9, 43.5, 21.5, 21.4, 21.3.
HR-FABMS Calcd for C26H37O12ClNa+ 599.1871, Found 599.1884. 1-Chloro-3,6-
dioxa-8-octyl 2,4,6-tri-O-acetyl-3-O-p-methoxybenzyl-b-D-galactoside (16.0 g, 27.7
mmol) was dissolved in a mixture (300 mL) of CH3CN and water (9/1). With ice
water bath cooling, CAN (38.0 g, 69.4 mmol) was added to the reaction vessel
portionwise during 2 h. After the reaction was kept for two more hours, the solvents
were partially removed in vacuo and water was added followed by extraction with
chloroform three times. The combined organic phase was washed with NaHCO3 and
brine. The solution was dried over anhydrous Na2SO4 and concentrated in vacuo to
give an oil, which was purified by flash chromatography to give 17 (10.3 g, 81%)
1
yield. H NMR (500 MHz, CDCl3) d 2.03 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.14 (s,
3H, OAc), 3.59–3.73 (m, 12H), 3.83 (m, 1H, H-3), 3.93 (m, 1H, H-5), 4.12 (m, 2H,
H-6a.b), 4.48 (d, J = 7.8 Hz, 1H, H-1), 4.95 (dd, J1 = 10.0 Hz, J2 = 7.8 Hz, 1H, H-2),
5.29 (d, J = 3.6 Hz, 1H, H-4). 13C NMR (125.7 MHz, CDCl3) d 171.7, 171.6, 171.2,
101.7, 73.2, 72.0, 71.8, 71.7, 71.3 (m), 71.1, 70.4, 69.6, 62.6, 43.4, 21.6, 21.4, 21.3.
HR-FABMS Calcd for C18H29O11ClNa+ 479.1296, Found 479.1315.
1-Azido-3,6-dioxa-8-octyl O-(2,3,4,6-tetra-O-benzyl-2-D-galactopyranolsyl)-
(1 ! 3)-2,4,6-tri-O-acetyl -D-galactopyranoside (19). Under an atmosphere of ni-
trogen, to a flame-dried 500 mL 3-neck flask equipped with an addition funnel, was
added 17 (10.0 g, 21.9 mmol), methyl 2,3,4,6-tetra-O-benzyl-1-thio-b-D-galactopyrano-
˚
side (18) (25.1 g, 44 mmol), 2,6-di-t-butyl 4-methyl pyridine (6.8 g, 33 mmol), 4 A
MS (10 g) and absolute ether (300 mL). After the mixture was stirred for 1 h, a
solution of methyl triflate (7.2 mL) in ether (50 mL) was added dropwise during 36 h.
After 48 h, the acceptor was completely consumed. The reaction mixture was filtered
through a celite pad and washed with ether. The filtrate was concentrated under
reduced pressure and chromatographed eluting with ethyl acetate and hexane (4/1 and
1/1) to give the disaccharide 1-chloro-3,6-dioxa-8-octyl O-(2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl)-(1 ! 3)-2,4,6-tri-O-acetyl-b-D-galactopyranoside (18.1 g, 84%) as a
1
coloress oil. H NMR (500 MHz, CDCl3) d 1.83 (s, 3H, OAc), 1.97 (s, 3H, OAc), 2.07
(s, 3H, OAc), 3.53 (d, J = 6.5 Hz, 2H, PhCH2 ꢁ), 3.62–3.76 (m, 12H), 3.84 (m, 2H),
3.88 (dd, J1 = 3.5 Hz, J2 = 10.5 Hz, 1H, H-2’), 3.95, (m, 2H, H-5, H-5’), 4.02 (dd,
J1 = 3.0 Hz, J2 = 9.5, 1H, H-3), 4.10–4.93 (m, 9H, 2PhCH2 ꢁ, 2H-6, 2H-6’, H-1), 5.10
(d, J = 3.5 Hz, 1H, H-1’), 5.21 (dd, J1 = 8.0 Hz, J2 = 10.0, 1H, H-2), 5.47 (d, J = 3.0
Hz, 1H, H-4), 7.26–7.39 (m, 20H, ArH). 13C NMR (125.7 MHz, CDCl3) d 171.2,
171.1, 169.9, 139.4, 139.3 (m), 138.8, 129.1 (m), 128.9, 128.8 (m), 128.7, 128.4 (m),

368
Wang et al.
128.3, 128.2 (m), 128.1, 102.2, 95.3, 79.2, 76.5, 76.0, 75.5, 74.3, 73.9 (m), 73.2, 72.1,
71.8, 71.4 (m), 71.0, 70.8, 70.5, 69.6, 69.4, 65.8, 62.6, 43.5, 21.6, 21.5, 21.2. HR-
FABMS Calcd for C52H63O16ClNa+ 1001.3702, Found 1001.3719. Sodium azide (0.68
g, 10.42 mmol) was added to a solution of 1-chloro-3,6-dioxa-8-octyl-O-(2,3,4,6-tetra-
O-benzyl-a-D-galactopyranosyl)-(1 ! 3)-O-(2,4,6-tri-O-acetyl-b-D-galactopyranoside)
(5.10 g, 5.21 mmol) in N,N-dimethylformamide (50 mL). The mixture was stirred
overnight at 80°C then cooled to room temperature. Chloroform (100 mL) was added
and the mixture was washed with water (30 mL) and brine (30 mL), dried with
Na2SO4, filtered and concentrated. The residue was chromatographed on silica gel
(eluent: hexane/ethyl acetate: 2/1, 1/1, sequentially) to give 19 (4.90 g, 95 %) as a pale
1
yellow oil. H NMR (500 MHz, CDCl3) d 1.83 (s, 3H, OAc), 1.98 (s, 3H, OAc), 2.07
(s, 3H, OAc), 3.87 (t, J = 4.5 Hz, 2H, -CH2N3), 3.54 (d, J = 6.5 Hz, 2H,), 3.64–3.74
(m, 10H), 3.84 (m, 2H), 3.89 (dd, J1 = 3.5 Hz, J2 = 10.5, 1H, H-2’), 3.95 (m, 2H, H-5,
H-5’), 4.02 (dd, J1 = 3.0 Hz, J2 = 9.5, 1H, H-3), 4.10–4.95 (m, 9H, 2PhCH2 ꢁ, 2H-6,
2H-6’, H-1), 5.10 (d, J = 3.5 Hz, 1H, H-1’), 5.21 (dd, J1 = 8.0 Hz, J2 = 10.0, 1H, H-2),
5.47 (d, J = 3.0 Hz, 1H, H-4’), 7.25–7.39 (m, 20H, ArH).13C NMR (125.7 MHz,
CDCl3) d 171.2, 171.1, 169.9, 139.5, 139.4, 139.3, 138.8, 129.1 (m), 128.9, 128.8 (m),
128.7, 128.4 (m), 128.3, 128.2 (m), 128.1, 102.3, 95.3, 79.2, 78.1, 76.5, 76.0, 75.5,
74.2, 73.9 (m), 73.2, 71.8, 71.4 (m), 71.0, 70.8, 70.7, 70.5, 69.7, 69.4, 65.8, 62.6, 51.4,
21.6, 21.5, 21.2. HR-FABMS Calcd for C52H63N3O16Na+ 1008.4106, Found
1008.4158.
Bis{N,N’-[O-(2,3,4,6-Tetra-O-benzyl- -D-galactopyranosyl)-(1 ! 3)-O-(2,3,6-
tri-O-acetyl-b-D-galactopyranosyl)-(1 ! 8)-3,6-dioxaoctyl]}terephthalamide
(21). A solution of 19 (0.30g, 0.31 mmol) in methanol (10 ml) was hydrogenated
(4 atm) at room temperature with platinum (IV) oxide hydrate (40 mg) as the catalyst.
After 1 h, the TLC showed that all starting material had been consumed. The reaction
mixture was diluted with methanol (30 mL), filtered and concentrated under reduced
pressure to give a white solid. The solid was dissolved in anhydrous dichloromethane
(10 mL) and triethylamine (87 mL, 0.62 mmol) was added. The reaction mixture was
then cooled to ꢁ 30°C. Terephthaloyl chloride (20, 24 mg, 0.12 mmol) was added
dropwise to the stirred mixture at ꢁ 30°C. Stirring was continued for 1 h at the same
temperature and the mixture was kept stirring for 12 h. Chloroform (30 mL) was added,
and the mixture was washed successively with 1M aqueous sodium bicarbonate (20
mL) , water (20 mL) and brine (20 mL) at room temperature. The organic layer was
dried with sodium sulfate and the solvents were evaporated in vacuo. The residue was
chromatographed on silica gel (eluent: chloroform/methanol: 40/1) to afford 21 (0.21g,
66% from 19, 88% based on terephthaloyl chloride 20) as a white foamy solid.1H NMR
(300 MHz, CDCl3) d 1.79 (s, 6H, OAc), 1.96 (s, 6H, OAc), 2.05 (s, 6H, OAc), 3.51–
3.73 (m, 28H), 3.79–4.09 (m, 16H), 4.34–4.89 (m, 18H, 4PhCH2 ꢁ, 4H-6, 4H-6’, 2H-
1), 5.07 (d, J = 3.3, 2H, 2H-1’), 5.19 (dd, J1 = 8.4 Hz, J2 = 10.2 Hz, 2H, 2H-2), 5.45
(d, J = 2.4 Hz, 2H, 2H-4), 7.20–7.40 (m, 40H, ArH), 7.88 (s, 4H, ArH). 13C NMR
(75.5 MHz, CDCl3) d 170.4, 170.3, 169.3, 166.5, 138.7, 138.6, 138.1, 137.0, 128.3,
128.2, 128.1, 127.9. 127.6, 127.5, 127.4, 127.3, 101.5, 94.7, 78.5, 75.7, 75.7, 75.3, 74.7,
73.5, 73.2, 72.5, 71.1, 70.5, 70.2, 70.1, 70.0, 69.8, 69.6, 69.0, 68.7, 65.1, 61.8, 39.8,
20.8, 20.7, 20.4. HR-FABMS Calcd for C112H132N2O34Na+ 2071.8559, Found
2071.8535.

FAC/MS: Effective Method for Kd Determination
369
1,4-Bis{N-[O- -D-galactopyranosyl-(1 ! 3)-O-b-D-galactopyranosyl]-(1 ! 8)-
3,6-dioxa-octyl]carbamido}benzene (22). A solution of 21 (0.15g, 0.073 mmol) in
methanol (15 mL) and acetic acid (1 mL) was hydrogenated (4 atm) at room
temperature with palladium hydroxide on carbon (20%) (20 mg) as catalyst for 24 h. The
reaction mixture was diluted with methanol (60 mL), filtered through a pad
of Celite
and concentrated to give a debenzylated residue. The residue was dissolved in absolute
methanol (100 mL), sodium methoxide was added and the pH of the solution was
adjusted to ca. 9. The reaction mixture was left stirring at room temperature overnight
and precipitated material was dissolved in the minimum amount of water, neutralized
with Dowex 50WX2-100 (H+) resin, filtered and washed with methanol. The solvents
were removed in vacuo. Purification by Bio-Gel (P2) column afforded the product 22
(0.65g, 83%) as a white solid. 1H NMR (500 MHz, D2O) d 3.42–3.54 (m, 34H), 3.62 (m,
2H, 2H-5’), 3.68 (dd, J1 = 3.6 Hz, J2 = 10.4 Hz, 2H, 2H-2’), 3.75 (dd, J1 = 3.1 Hz,
J2 = 10.4 Hz, 2H, 2H-3’), 3.81 (d, J = 3.1 Hz, 2H, 2H-4’), 3.85 (m, 2H, 2H-5), 3.99 (m,
4H, 2H-4, 2H-6a), 4.25 (d, J = 8.0 Hz, 2H, 2H-1), 4.96 (d, J = 3.6 Hz, 2H, 2H-1’), 7.68
(s, 4H, ArH). 13C NMR (125.7 MHz, D2O) d 170.0, 136.6, 127.4, 102.5, 95.2, 77.1, 74.8,
70.7, 69.6, 69.5, 69.3, 69.2, 69.1, 69.0, 68.7, 68.5, 68.1, 64.7, 60.8, 39.5. HR-FABMS
Calcd for C44H72N2O28Na+ 1099.4169, Found 1069.4179.
1,3,5-Tris{N-[O-(2,3,4,6-tetra-O-benzyl- -D-galactopyranosyl)-(1 ! 3)-O-2,4,6-
tri-O-acetyl- -D-galactopyranosyl)-(1 ! 8)-3,6-dioxaoctyl] carbanmido}benzene
(23). A solution of 19 (0.38g, 0.39 mmol) in methanol (20 mL) was hydrogenated
(4 atm) at room with platinum (IV) oxide hydrate (50 mg) as the catalyst. After 1 h,
TLC showed the starting material had been consumed. The reaction mixture was
diluted with methanol (40 mL), filtered and concentated under reduced pressure to give
a white solid. The solid was dissolved in anhydrous dichloromethane (10 mL) and
triethylamine (0.16 ml, 1.17 mmol) was added. The reaction mixture was then cooled
down to ꢁ 30°C. 1,3,5-benzentricarbonyl trichloride (25 mg, 0.096 mmol) was added
dropwise to the stirred mixture at ꢁ 30°C. Stirring was continued for 1 h at the same
temperature and left stirring for 12 h at room temperature. Chloroform (30 ml) was
added and the mixture washed successively with 0.5 N HCl and 1M aqueous sodium
bicarbonate (20 mL), water (20 mL) and brine (20 mL). The solution was dried with
sodium sulfate and the solvents were evaporated in vacuo. The residue was
chromatographed on silica gel (eluent: chloroform/methanol: 40/1) to afford 23
(0.32g, 62% from 19, 83% based on 1,3,5-benzentricarbonyl trichloride 20) as a white
1
foamy solid. H NMR (300 MHz, CDCl3) d 1.81 (s, 9H, OAc), 1.95 (s, 9H, OAc), 2.04
(s, 9H, OAc), 3.51 (d, J = 6.6 Hz, 6H), 3.60–3.71 (m, 36H), 3.79–4.05 (m, 24H),
4.36–4.85 (m, 27H, 6PhCH2 ꢁ, 6H-6, 6H-6’, 3H-1), 5.07 (d, J = 3.6 Hz, 3H, H-1’),
5.16 (dd, J1 = 7.8Hz, J2 = 9.9 Hz, 3H, H-2), 5.44 (d, J = 3.0 Hz, 3H, H-4), 7.24–7.38
(m, 60H, ArH), 8.39 (s, 3H, ArH). 13C NMR (75.5 MHz, CDCl3) d 170.4, 170.3, 169.1,
165.7, 138.7, 138.6, 138.1, 135.2, 128.3–127.4, 101.5, 94.7, 78.4, 75.7, 75.3, 74.7,
73.5, 73.2, 72.6, 71.0, 70.6, 70.3, 70.1, 69.7, 69.6, 68.9, 68.7, 65.1, 61.8, 40.1, 20.8,
20.7, 20.4.
1,3,5-Tris{N-[O- -D-galactopyranosyl-(1 ! 3)-O- -D-galactopyranosyl-(1 ! 8)-
3,6-dioxa-octyl]carbamido}benzene (24). A solution of 23 (0.24g, 0.079 mmol) in
methanol (20 mL) and acetic acid (1 mL) was hydrogenated (4 atm) at room with

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Wang et al.
palladium hydroxide on carbon (5%) (80 mg) for 24 h. The reaction mixture was
diluted with methanol (60 mL), filtered through a pad of Celite, and concentrated to
give a debenzylated residue. The residue was dissolved in absolute methanol (100 mL).
Sodium methoxide was added and the pH of the solution was adjusted to ca. 9. The
reaction mixture was left stirring at room temperature overnight and precipitated
material was dissolved in the minimum amount of water. The reaction was then
neutralized with Dowex 50WX2-100 (H+) resin, filtered, and washed with methanol.
The solvents were removed in vacuo. Purification by Bio-Gel (P2) column afforded the
1
product 24 (0.10g, 80%) as a white solid. H NMR (500 MHz, D2O) d 3.44–3.54 (m,
51H), 3.65 (m, 3H, 3H-5’), 3.70 (dd, J1 = 3.6 Hz, J2 = 10.2 Hz, 3H, 3H-2’), 3.76 (dd,
J1 = 3.2 Hz, J2 = 10.2 Hz, 3H, 3H-3’), 3.82 (d, J = 3.2 Hz, 3H, 3H-4’), 3.85 (m, 3H,
3H-5), 3.99 (m, 6H, 3H-4, 3H-6a), 4.26 (d, J = 8.0 Hz, 3H, 3H-1), 4.96 (d, J = 3.6 Hz,
3H, 3H-1’), 8.14 (s, 3H, ArH). 13C NMR (125.7 MHz, D2O) d 168.9, 134.8, 128.9,
102.5, 95.2, 77.1, 74.6, 70.7, 69.9, 69.5, 69.3, 69.2, 69.1, 69.0, 68.7, 68.5, 68.1, 64.7,
60.8, 39.6. HR-FABMS Calcd for C63H105N3O42Na+ 1598.6070, Found 1598.5996.
Enzymatic synthesis of the -Gal trivalent compound 26. Trilactose 25 (4.3
mg, 3.8 mmol), UDP-Gal (7.7 mg, 12.3 mmol) and 2 mL of alkaline phosphatase (1 U/
mL) were added to a 1.5-mL solution of a1,3-Gal T (from calf thymus, 40 mU/mL in
30 mM sodium cacodylate, 20 mM MnCl2, 0.1 % Triton X-100, pH 6.5). The mixture
was incubated with gentle rotation at room temperature for 4 days with addition of
UDP-Gal (2 mg) and a1,3-Gal T (600 mL) every two days. The reaction mixture was
clarified by centrifugation (Microsepconcentrate, NMWL cut of 30 kDa) and the
sample was purified on a Bio-Gel (P2) column. Further purification was carried out by
loading the crude product onto two sequential C18 Sep-Pak cartridges. To remove
Triton-X-100, the cartridges were washed with water (80 mL) and 50% aqueous MeOH
(30 mL). The eluate was concentrated, redissolved in water, filtered through a Miller-
1
GV 0.22 mM filter and lyophilized to yield 26 (3.9 mg, 64%) as a white solid: H NMR
(600 MHz, D2O) d 3.33 (dd, J1 = 7.8 Hz, J2 = 9.3 Hz, 3H-2 of b-Glc), 3.60–3.82 (m,
36H), 3.86 (dd, J1 = 3.8 Hz, J2 = 10.4 Hz, 3H-2 of a-Gal), 3.95 (dd, J1 = 3.6 Hz,
J2 = 10.4 Hz, 3H-3 of a-Gal), 3.99 (dd, J1 = 2.0 Hz, J2 = 12.6 Hz, 3H-3 of b-Gal),
4.02 (d, J = 3.3 Hz, 3H-4 of b-Glc), 4.18 (d, J = 3.3 Hz, 3H-4 of b-Gal), 4.20 (d,
J = 4.0 Hz, 3H-4 of a-Gal), 4.21 (d, J = 11.2 Hz, 3Ha of NO2-Tris), 4.46 (d, J = 11.2
Hz, 3Hb of NO2-Tris), 4.51 (d, J = 7.8 Hz, 3H-1 of b-Glc), ), 4.52 (d, J = 7.8 Hz, 3H-1
of b-Gal), ), 5.15 (d, J = 3.8 Hz, 3H-1 of a-Gal). HR-ESMS: Calcd for C58-
H99NO50Na+ 1632.5132, Found 1632.5128.
Enzymatic synthesis of -Gal terminated N-linked oligosaccharide 28. Tyro-
sinamide N-linked oligosaccharide 27 (7 mg, 3.0 mmol), UDP-Gal (0.109 g, 60 mmol)
was added to a 3-mL solution of bovine a1,3-galactosyltransferase[42] (2.8 U/3mL in
100 mM Tris buffer, pH 7.0, 500 mM NaCl, 30 mM MnCl2, 1% BSA, 50 U of alkaline
phosphatase). The mixture was incubated with gentle shaking at room temperature for 7
days. The reaction mixture was filtered through glass wool and then ultrafiltered
(NMWL 10,000) to remove the protein. The filtrate was lyophilized and purified on a
Bio-Gel (P4) column with water as eluent. Further purification was carried out using
semi-preparative HPLC. The sample was dissolved in water and applied to a
1 ꢃ 25 cm semi-preparative C18 reverse phase (RP) HPLC column and eluted

FAC/MS: Effective Method for Kd Determination
371
isocratically with 12% acetonitrile at 1 mL/min. The eluting peaks detected by UV
absorbance at 280 nm (0.3 aufs) were pooled separately and lyophilized (Figure 4). The
major peak (1) corresponded to the desired a-Gal terminated N-linked oligosaccharide
1
28 (5.2 mg, 61%) as a white solid. H NMR (600 MHz, D2O) d 1.34 (s, br, 9H, Boc),
1.98 (s, 3H, 1-NAc), 2.05 (s, br, 6H, 5,5’-NAc), 2.08 (s, 3H, 7-NAc), 2.09 (s, 3H, 2-
NAc), 2.82 (m, 1H, PhCH2 ꢁ), 3.00 (m, 1H, PhCH2 ꢁ), 3.49 (t, J = 9.5 Hz, 1H,
PhCH2CH-), 3.56–4.06 (m), 4.12 (d, J = 3.1 Hz, 1H), 4.18–4.25 (m, 8H), 4.53–4.56
(m, 4H, H-1 of 6, 8, 6’, 7), 4.57 (d, J = 7.1 Hz, 1H, H-1 of 5’), 4.58 (d, J = 7.8 Hz, 1H,
H-1 of 5), 4.62 (d, J = 8.0 Hz, 1H, H-1 of 2), 4.74 (overlap with H2O, 1H, H-1 of b-
Man), 4.93 (s, 1H, H-1 of 4’), 5.02 (d, J = 9.7 Hz, 1H, H-1 of 1), 5.12 (s, 1H, H-1 of
4), 5.15 (d, J = 3.6 Hz, 3H, H-1 of G, G’, G@). ESMS: m/z 2777 (MNa+), 1400
(M + 2Na+).
ACKNOWLEDGMENTS
This work was supported by grants from the Natural Sciences and Engineering
Research Council of Canada (O. Hindsgaul and M. M. Palcic); NIH Foundation
(GM 54074 & GM 54073) and NSF (BES-9728366) of the United States (P. G.
Wang). B. Zhang was supported by a fellowship from the Alberta Heritage Foun-
dation for Medical Research. We thank Prof. P. Hultin, University of Manitoba, for
compound 25.
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