CD22-Antagonists with nanomolar potency: The synergistic effect of hydrophobic groups at C-2 and C-9 of sialic acid scaffold

Article abstract of DOI:10.1016/j.bmc.2011.01.060

In earlier studies, we identified the C-9 amido derivative 1 (9-(4′-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) and the C-9 amino derivative 2 (9-(4′-hydroxy-4-biphenyl)methylamino-9-deoxy- Neu5Gcα2-6GalOMP) have the most promising affinity for

Full text of DOI:10.1016/j.bmc.2011.01.060

Bioorganic & Medicinal Chemistry 19 (2011) 1966–1971
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
CD22-Antagonists with nanomolar potency: The synergistic effect
of hydrophobic groups at C-2 and C-9 of sialic acid scaffold
Hajjaj H. M. Abdu-Allah a, , Kozo Watanabe b,c,d, Gladys C. Completo e, Magesh Sadagopan f,
⇑
Koji Hayashizaki b,c, Chiaki Takaku b,c, Taichi Tamanaka b,c, Hiromu Takematsu g, Yasunori Kozutsumi g,
James C. Paulson e, Takeshi Tsubata b,c,d, Hiromune Ando a,f, Hideharu Ishida a, Makoto Kiso a,f,
⇑
a Department of Applied Bio-organic Chemistry, Faculty of Applied Biological Sciences, The United Graduate School of Agricultural Sciences, Gifu University, Gifu 501-1193, Japan
b Laboratory of Immunology, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Japan
c Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Japan
d CREST, Science and Technology Agency (JST), Saitama, Japan
e Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA
f Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
g Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Japan
a r t i c l e i n f o
a b s t r a c t
In earlier studies, we identified the C-9 amido derivative 1 (9-(40-hydroxy-4-biphenyl)acetamido-9-
Article history:
deoxy-Neu5Gc
9-deoxy-Neu5Gc
a
2-6GalOMP) and the C-9 amino derivative 2 (9-(40-hydroxy-4-biphenyl)methylamino-
Received 19 November 2010
Revised 26 January 2011
Accepted 28 January 2011
Available online 2 February 2011
a
2-6GalOMP) have the most promising affinity for mouse CD22 and human CD22,
respectively. Replacing the subterminal galactose residue (2-6Gal-OMP) of 1 with benzyl (5) or biphe-
nylmethyl (6) as aglycone led to even higher potency for mCD22. In this study, both compounds showed
improved potency and selectivity for CD22 (IC50 70 nM) and 712-fold more selective for CD22 than for
MAG. The corresponding derivatives of 2, compounds 8 and 9, showed comparable activity to 2 but lower
potency and selectivity than 5 and 6. Although compounds 5–9 are simple and small molecular weight
antagonists, they showed much high potency and selectivity than the corresponding compounds having
Keywords:
Benzyl and biphenylmethyl sialosides
CD22
Hydrophobic interaction
Selectivity
MAG
a
2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6Gal-
OMP residues of 1 and 2 are not critical for binding process and could be replaced with hydrophobic
non-carbohydrate moieties. The data presented herein has significant implications for the design and dis-
covery of next-generation CD22-antagonists.
ELISA
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
interact with B cells (e.g., T cells, trans ligands). Interactions of
CD22 with cis or trans ligands regulate aspects of B cell activa-
Advances in the functional understanding of carbohydrate–
protein interactions have enabled the development of a new class
of small-molecule drugs, known as glycomimetics. These com-
pounds mimic the bioactive function of carbohydrates and address
the drawbacks of carbohydrate leads, namely their low activity and
insufficient drug-like properties.1
CD22 (siglec-2) is the best characterized member of siglec
family.2 It is an accessory molecule of the B-cell receptor complex
(BCR) that exhibits both positive and negative effects on receptor
signaling. The carbohydrate ligand recognized by CD22 is the
tion, proliferation and development. Therefore, CD22 is a regula-
tory protein that prevents the overactivation of the immune
system and the development of autoimmune diseases.3 Our cur-
rent understanding of the biological functions of CD22 is mostly
based on in vitro experiments. The present challenge will be to
transfer this knowledge to in vivo studies, in order to learn more
about their role in regulating immune responses. To face this
challenge, high affinity ligands suitable for such studies are re-
quired. In addition CD22 antagonists with sufficient avidity could
find several applications, for example, modulation of the immune
response4 and targeting device for treatment of B cell related
diseases.5 Chen et al., 2010.
sequence Siaa2-6Galb1-4GlcNAc found on both neighboring
glycoconjugate of the same cell (cis ligands) and on cells that
Recently,6 we have reported the dramatic improvement of
binding affinity for CD22 achieved by the modifications at C-9 of
⇑
Corresponding authors. Present address: Department of pharmaceutical
Neu5Gc
1 (9-(40-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gc
lOMP) and the 9-amino derivative 2 (9-(40-hydroxy-4-biphe-
nyl)methylamino-9-deoxy-Neu5Gc 2-6GalOMP) which exhibited
a
2-6GalOMP core as exemplified by the 9-amido derivative
Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
(H.H.M.A). Tel.: +81 (0) 58 293 2918, fax: +81 (0) 58 293 2840 (M.K.).
E-mail addresses: moazhajjaj@yahoo.com (H.H.M. Abdu-Allah), Kiso@gifu-
u.ac.jp (M. Kiso).
a
2-6Ga-
a
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.060

H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. 19 (2011) 1966–1971
1967
the highest potency for mouse CD22 (mCD22) and human CD22
(hCD22), respectively, (Fig. 1). Our previous docking studies
revealed that C-9 amido or amino sialic acid derivatives linked with
2-6GalOMP moiety form extra interactions as compared with
reference compound, which could account for their increased
binding affinities for both hCD22 and mCD22.6
More recently, we have found that replacing the subterminal
galactose residue of 1 (2-6GalOMP) with benzyl or biphenylm-
ethyl as aglycone at the C-2 of sialic acid scaffold led to higher
affinity for mCD22 (compounds 5 and 6; Scheme 1).7 The further
analysis of binding affinity of the tested compounds guided that
2-6GalOMP group which is independent from the core sialic acid,
could be replaced with more flexible hydrophobic groups. The
hydrophobicity of benzyl or biphenylmethyl could compensate
the desolvation penalty fetched by 2-6Gal-OMP (unfavorable
desolvation enthalpy of OH groups in the 2-6Gal) and also gain
in the entropy (the change in solvation entropy is more favor-
able if the surfaces are more hydrophobic) can make the binding
affinity more favorable.
Sequence comparison of Siglecs and inspection of the 2-3-sialyl-
lactose/SnD1 crystal structure suggested that -sialosides with
a
modified substituents in the glycerol side chain could yield in-
creased binding affinities.8–10 Moreover, such studies indicated
that these compounds would be likely to display enhanced speci-
ficity to individual family members and could potentially aid in
the dissection of Siglec function.10
In continuation of our interest in investigating the molecular
basis of the interaction of CD22 with sialosides, we report herein
the synthesis of the new compounds 7–9 and the determination
of the binding affinity of compounds 5–9 for CD22 and MAG. Man-
ual docking and molecular dynamics simulations were carried out
to investigate the structural basis of the observed affinity.
2. Results and discussion
This work describes the novel small molecule hydrophobic
sialosides as selective antagonists for the ligand binding domain
of CD22.
HO
O
NH
HN
COOH
OH
COOH
OH
HO
HO
GcHN
HO
GcHN
O
O
O
O
HO
HO
HO
HO
O
O
O
O
HO
HO
HO
HO
2
1
OMe
OMe
Figure 1. Structures of the 9-amido (1) and 9-amino (2) sialosides.6
HO
O
O
N
O
O
O
NH
HO
HOCH2COHN
COOH
O
OH
(i)
O
HO
HO
Ar
5 Ar = phenyl
6 Ar = biphenyl
H2N
HO
HOCH2COHN
F
COOH
O
OH
O
HO
O
N
O
O
HO
Ar
O
O
3 Ar = phenyl
4 Ar = biphenyl
HO
NH
OH
COOH
HO
O
O
HOCH2COHN
HO
7
)
ii
(
CHO
HO
NH
OH COOH
HO
O
O
HOCH2COHN
OH
Ar
8 Ar = phenyl
9 Ar = biphenyl
Scheme 1. Synthesis of 9-amido (5–7) and 9-amino (8 and 9) benzyl and biphenylmethyl sialosides. Reagents and conditions: (i) NaHCO3, MeCN/H2O, 75%; (ii) NaBH3CN,
AcOH, MeOH, rt, 24 h, 68%.

1968
H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. 19 (2011) 1966–1971
Table 3
2.1. Chemistry
Comparison of inhibitory potentials of the synthetic sialosides to CD22 and MAG
As outlined in our preceding communication7 we have achieved
the syntheses of benzyl 3,5,9-trideoxy-5-glycolamido-9-(40-hydro-
xy-4-biphenyl)acetamido-D-glycero-a-D-galacto-2-nonulopyranos-
Compd No.
Activity
M)
Selectivity
hCD22 IC50
(l
MAG IC50 (lM)
1
2
5
6
8
9
3.27 0.62
3.29 0.33
0.24 0.05
0.38 0.00
2.75 1.3
136 27
37.4 3.1
171 35
164 11
9.27 1.3
16.6 5.2
41.5
11.4
712.5
431.5
3.4
idonic acid (5) and the corresponding biphenylmethyl sialoside (6)
by acylation of the 9-amino intermediates (3 and 4), respectively,
with N-hydroxysuccinimide ester of 40-hydroxybiphenyl-4-acetic
acid (Scheme 1). Similarly compound 7 was prepared by acylation
of 3 with the succinimidyl ester6 of 40-fluorobiphenyl-4-acetic
acid.11 Based on the higher affinity of 9-aminosialoside 2 for
hCD22 than 9-amidosialosides 16 and encouraged by the promis-
ing activity of compounds 5 and 6; the corresponding 9-amino
analogues (8 and 9) were prepared. They were synthesized by
reductive alkylation of 3 and 4 with 40-hydroxybiphenyl-4-carbal-
dehyde6 (Scheme 1).
2.24 0.28
7.4
The selectivity of each sialoside was determined. They were calculated by dividing
the IC50 value of each compound for MAG by the IC50 value of the same compound
for CD22.
are the most potent antagonists for hCD22 (IC50 130 nM and
70 nM). In case of hCD22, biphenyl methyl sialoside (6) is more po-
tent than benzyl sialoside (5), while in case of mCD22, benzyl sialo-
side is more potent. This result may be due to steric interaction
with the binding site as will be discussed in the molecular model-
ing study. Unexpectedly, and in contrast with the improved po-
tency of the analogs of 1, the benzyl and biphenylmethyl analogs
of 2 (8 and 9) did not show any improvement of binding affinity
(Table 2). They are nearly equipotent to 2. Since the analogs of 1
and 2 are different only in the substituent at C-9, it seems that
the flexibility of amino group is responsible for such differences
in binding affinity. Therefore, the greater flexibility of compounds
8 and 9 may result in entropic penalties upon binding. Alterna-
tively, the flexibility may constrain multivalent interaction with
CD22, while the 9-amido group of 5 and 6 is more rigid giving
the correct orientation for multivalent interaction. Despite having
a simpler structure the target compounds have either higher (5
and 6) or comparable (8 and 9) potency to more complex parent
structures 1 and 2. These sialic acid derivatives combine the bene-
ficial modifications at C-2 and C-9 positions in one molecule and
are supposed to have improved pharmacokinetic properties due
to the presence of aromatic moieties, a lower molecular weight,
and a reduced number of polar hydroxy functions compared to
the parent compounds. Compound 5 and 6 are the most potent
inhibitors for hCD22 have ever been synthesized.
2.2. Biological assay
A competition enzyme-linked immunosorbent assay (ELISA)
based on a biotinylated form of 112 was used for measuring the
inhibitory potency of the synthesized sialosides. The results are
shown in Tables 1–3.
2.2.1. Comparison between the binding affinity of 9-amido (5
and 6) and 9-aminosialosides (8 and 9) for hCD22 and mCD22
The high affinity of 5 (IC50 100 nM, 38-fold higher potency) and
6 (IC50 190 nM, 20-fold higher potency) for mCD22 in comparison
with 1,7 prompted us to investigate their potencies for hCD22.
Accordingly, we found that both compounds 5 and 6 also showed
improved affinity for hCD22; 7 and 12-fold more potent than 1,
respectively. As observed for mCD22, these hydrophobic sialosides
Table 1
Inhibitory potencies of 9-amido analogs (5–7) for mCD22 and hCD22
Compd
Activity
mCD227
M)
hCD22
IC50 (lM)
IC50
(
l
rIPb
rIP
a
1
5
6
7
3.84 0.32
0.10 0.00
0.19 0.00
5.00 0.6
1
38.4
20.2
0.90 0.00
0.13 0.00
0.07 0.01
ND
1
7
12
2.2.2. Comparison between the binding affinity of the test
compounds for CD22 and MAG
0.77
At this stage of development of these potent inhibitors, it was
crucial to address the issue of specificity of the synthetic sialosides
for CD22. Therefore, appropriate control, a closely related Siglec-4
(myelin-associated glycoprotein, MAG), was included in the ELISA
experiments to ascertain if the inhibitors are really specific to
CD22.
a
Sialoside concentration which leads to 50% inhibition of binding. The values are
the mean SD of triplicates.
b
The rIP of each sialoside was calculated by dividing the IC50 of the reference
compound 1 by the IC50 of the compound of interest. The results in rIPs above 1.0
for derivatives binding better than the reference and rIPs below 1.0 for compounds
with a lower affinity than the reference. ND; not determined. The values for mCD22
and hCD22 cannot be directly compared.
Inhibitory potentials of the synthetic sialosides to CD22 and
MAG were determined by competitive ELISA (Table 3). The most
interesting results in our study are the high binding affinity and
selectivity of compounds 5 and 6 (Table 3). Both of them contain
the same hydrophobic moiety at C-9 and benzyl or biphenyl,
respectively, at C-2. Compound 5 is 712-fold more selective for
hCD22 than for MAG. Similarly compound 6 is 431-fold more
selective. Surprisingly, they are much more selective than the cor-
responding compounds containing a2-6 linkage; 1 and 2. They are
also much more potent and selective than 8 and 9 which contain 9-
amino instead of 9-amido.
On the other hand, binding studies on MAG revealed that benzyl
sialosides are 10-fold more potent than methyl sialosides.13 This
effect was rationalized by the results of STD-NMR investigations,
indicating a hydrophobic interaction with MAG.14 Moreover, chlo-
robenzamide was found to be the best substituent in the 9-posi-
tion.15 Accordingly, we can conclude that the main source of
Table 2
Inhibitory potencies of 9-amino analogs (8 and 9) for hCD22 and mCD22
Compd
Activitya
mCD22
M)
hCD22
M)
IC50
(l
rIPb
IC50
(
l
rIP
2
8
9
6.43 0.00
5.03 3.54
23.33 5.51
1
1.2
0.3
0.53 0.00
0.82 0.02
1.12 0.06
1
0.6
0.5
a
Sialoside concentration which leads to 50% inhibition of binding. The values are
the mean SD of triplicates.
b
The rIP of each sialoside was calculated by dividing the IC50 of the reference
compound 1 by the IC50 of the compound of interest. The results in rIPs above 1.0
for derivatives binding better than the reference and rIPs below 1.0 for compounds
with a lower affinity than the reference. ND; not determined. The values for mCD22
and hCD22 cannot be directly compared.

H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. 19 (2011) 1966–1971
1969
selectivity of 5 and 6 is the presence of 9-(40-biphenylacetamido)
which contributes significantly to the improved affinity as well
as selectivity.
2.2.3. The effect of replacing 40-hydroxy by 40-fluoro on the
binding affinity of compound 5
The promising activity and selectivity of 5 encouraged us to
study the effect of replacing the 40-hydroxy group with 40-fluoro.
Accordingly, compound 7 was synthesized and its binding for
CD22 was evaluated. The rationale for such strategy stems from
similarities between fluorine and oxygen in polarity and close isos-
teric relationship.16 Consequently, a F atom is considered as a good
substitute of a OH group. One important advantage of introducing
fluoride is to render the compound relatively resistant to metabolic
transformation due to the high energy of carbon–fluorine bond.16
Unexpectedly, we found that compound 7 has significantly lower
activity for CD22 (IC50 5 lM; 50-fold less potent than 5). Obviously,
a fluorine atom cannot donate hydrogen bonds, but it is electro-
negative and can accept them. This finding confirms our model
of binding to CD22 and the involvement of 40-hydroxy in H-bond
formation.6
Figure 2. View of the simulated representative structure of compound 5 bound to
the mCD22. Structures colored in grey are protein residues.
The solubility of the test compounds in aqueous media and
the reproducibility of the assay results under different condition
from different laboratories are thought to provide insight into
the interactions with the binding site and exclude the possibility
of micelle or multimeric formation as one possible mechanism of
improved activities of such compounds. Moreover, the lower
activity of compound 7 gives a good evidence for the depen-
dence of binding on structural feature and not on physical inter-
actions of molecules. In addition, closely related structures of
MAG antagonists have been reported and the improved activities
of such compounds were accounted for by increased lipophilicity
as rationalized by the results of trNOE NMR and STD NMR inves-
tigations.13–15
2.3. Molecular modeling
In order to investigate the structural basis of the observed affin-
ity and interpret the unexpected differences in the inhibition
potencies between 9-amido (5 and 6) and 9-amino (8 and 9) ana-
logs, manual docking and molecular dynamics simulations were
carried out against mCD22. The binding modes of the benzyl sialo-
sides (5 and 8) apparently found to be different with the previously
studied 2-6Gal-OMP binding mode.6
As can be seen from Figure 2, the benzyl portion of the repre-
sentative structures obtained during simulations of 5 is slightly ex-
posed out to solvent and partially interacts with the 9-
biphenylacetamido portion of the same compound. These attrac-
tive intra-molecular interactions could be induced dipole–induced
dipole (London) dispersion forces as both aromatic groups (C-2
phenyl and C-9 biphenyl of 5) are present in their proximities
(ꢀ3.1 Å to 5.0 Å). These momentary van der Waals attractions
could also be aided by the solvophobic effects, in which desolva-
tion can stabilize interactions of hydrophobic molecular surfaces.
Therefore, they can stabilize protein ligand complex.17 In connec-
tion with this, the differential activities of benzyl or biphenylmeth-
yl sialosides between the hCD22 and mCD22 can be inferred to
subtle amino acid differences in the C-9-biphenyl binding region
(Arginine in hCD22, Proline in mCD22).6
Figure 3. View of the simulated representative structure of compound 8 bound to
the mCDD22. Structures colored in grey are protein residues.
the intra-molecular interactions with the head and tail hydropho-
bic portions.
Previously reported crystal structures of the Siglec sialoadhesin
(SnD1), in the absence of ligand, and in complex with sialic acid
analogs, revealed that SnD1 undergoes very few structural changes
on ligand binding which unexpectedly can induce dimerization of
two SnD1 molecules.18 These results highlight the need to account
for the possibility of ligand induced dimerization when measuring
affinity.18 On the other hand, thermodynamic analysis of recently
developed MAG antagonists, having hydrophobic substituents at
C-2 of sialic acid moiety, revealed that the improved affinity pre-
dominantly results from an increased binding enthalpy and not
from an entropy gain.19 Hydrophobic interactions with the side
chains of Trp59, Tyr60, and Tyr69, lining the main hydrophobic
pocket was proposed through homology modeling and docking.19
Both of the above mentioned facts could not sufficiently ratio-
nalize our results; though second one look more promising. How-
ever, the sequence alignment of some member of siglecs showed
that the hydrophobic cavity of MAG is not relevant or not clearly
known in CD22 and Sn.10
On the other hand, C-2 benzyl and C-9 biphenyl of 7 (an amino
analogue of compound 5) are stay farther distance than those of 5
(ꢀ4.6 Å to 8.5 Å; Fig. 3). This could be due to the flexibility of amino
group, which can disturb the intra-molecular interactions de-
scribed above. At this point, we can speculate that the observed
differences in the activity of compounds 5–9 as compared to the
parent compounds (1 or 2) could be due to their ability to form
Based on our models of CD22,6 2-benzyl or biphenyl group are
slightly exposed to the solvent and also interacting the 9-biphenyl
group in a shallow active of CD22s. The proposed intra-molecular

1970
H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. 19 (2011) 1966–1971
interactions, between C-2 and C-9 substituents, could be better in
explaining the observed activity differences. Residues related to
hydrophobic cavity of MAG in the CD22s are not in same of posi-
tion or even close; if 2-benzyl of 5 takes similar conformation as
seen in MAG antagonists, it can severely affect the conserved
Arg-97 interaction in CD22s.
Taken together, the current experimental and computational
studies support our previous finding that the subterminal sugar
part (2-6Gal-OMP) could be replaced with non-carbohydrate moi-
eties. Particularly, groups with optimal hydrophobicity could be
exploited for improved binding affinity.
tion from water. The solvent system (ethyl acetate/methanol/
water/acetic acid 10:3:3:1) was used for following up the reaction.
1H NMR (CD3OD): d 7.60–7.45 (m, 4H, Ar-H), 7.40–7.10 (m, 9H,
Ar-H), 4.78 (d, J = 11.4 Hz, 1H, glycosidic CH2), 4.5 (d, J = 11.0 Hz,
1H, glycosidic CH2), 4.03 (s, 2H, glycolyl CH2CO), 3.96–3.92 (m,
1H, H8b), 3.82–3.67 (m, 4H, H4, H5, H6, H7), 3.57 (br s, 2H, biphCH2-
CONH), 3.37 (br d, J = 9.8 Hz, 1H, H9a), 3.20 (dd, J = 9.8, 13.7 Hz, 1H,
H
H
9b), 2.90 (dd, J = 4.6, 12.0 Hz, 1H, H3eq), 1.64 (t, J = 12.0 Hz, 1H,
3ax). 13C NMR (CD3OD): d 180.2, 178.9, 174.3, 169.7, 158.12,
150.5, 141.0, 140.0, 137.1, 135.4, 131.5, 129.2, 129.1, 128.9,
128.3, 127.5, 116.7, 116.6, 114.1, 102.1, 74.0, 72.3, 71.3, 69.5,
67.4, 62.6, 61.4, 44.0, 43.4, 42.7, 26.3. MALDI-TOF MS calcd for
C
32H35FN2O10Na (M+Na)+, 626.23; found 626.22.
3. Conclusion
4.2. General procedures for reductive alkylation
Structurally simplified and highly potent CD22-antagonists of
high selectivity were synthesized. The improved affinity of the
target compounds (5–9) might be due to desolvation and intra-
molecular interactions of hydrophobic groups at C-2 and C-9 or
due to dimerization of CD22 molecules. The higher binding affinity
exhibited by the more rigid 9-amido derivatives (5 and 6) in com-
parison with the flexible 9-amino derivatives (8 and 9) may be due
to the stronger intra-molecular forces between the hydrophobic
moieties at C-2 and C-9. We are encouraged by these promising
results and are moving forward regarding the optimization of the
substituent at C-2 of sialic acid scaffold for further improvement
of pharmacodynamic and pharmacokinetic parameters.
To a mixture of the amine (3 or 4, 0.084 mmol) and 40-hydrox-
ybiphenyl-4-carbaldehyde6 (8.5 mg, 0.043 mmol) in dry MeOH
(3 mL) was added acetic acid (0.1 mL), followed by NaBH3CN
(1.0 M in THF, 60 lL, 0.060 mmol). After stirring at rt for 24 h,
the reaction mixture was concentrated, reconstituted into water
and loaded on a silica reversed phase column pre-equilibrated in
water. The compounds were eluted with a gradient of methanol–
water (0:1–60:100) to afford the title compounds 7 or 8 in about
68% yield as a white fluffy solid after a final lyophilization from
water. The solvent system (ethyl acetate/methanol/water/acetic
acid 10:3:3:1) was used for following up the reaction.
4.2.1. Benzyl 3,5,9-trideoxy-5-glycolamido-9-(40-hydroxy-4-
4. Experimental
4.1. Synthesis
biphenyl)methylamino-D-glycero-a-D-galacto-2-
nonulopyranosidonic acid (8)
1H NMR (CD3OD): d 7.62–7.21 (m, 11H, Ar-H), 6.86–6.83 (m, 2H,
Ar-H), 4.81 (d, J = 11.0 Hz, 1H, glycosidic CH2), 4.71 (d, J = 11.0 Hz,
1H, glycosidic CH2), 4.24–4.16 (m, 3H, H8, glycolyl CH2CO), 4.05
(s, 2H, biphCH2NH), 3.89–3.85 (m, 1H, H4), 3.75–3.69 (m, 2H, H5,
H6), 3.40 (d d, J = 8.9, 1.8 Hz, 1H, H7), 3.36 (dd, J = 2.8, 12.7 Hz,
4.1.1. General information
1H and 13C NMR spectra were recorded on JEOL JNM-EX-400
(400 MHz) or JEOL JNM-ECA-500 (600 MHz). Chemical shifts are
expressed in ppm (d) relative to the signal of Me4Si. MALDI-TOF
MS spectra were recorded in positive ion mode on a Bruker Auto-
1H,
H9a), 2.96 (br d, J = 12.7 Hz, 1H, H9b), 2.93 (dd, J = 4.8,
flex with the use of a-cyano-4-hydroxycinnamic acid (CHCA) as a
12.4 Hz, 1H, H3eq), 1.66 (t, J = 12.4 Hz, 1H, H3ax). MALDI-TOF MS
calcd for C31H37N2O10 (M+H)+, 597.24; found 597.24.
matrix. Solvents and reagents were used as received from commer-
cial distributors without further purification. Anhydrous reactions
were conducted under an argon atmosphere. TLC analysis was car-
ried out on Merck TLC (Silica Gel 60F254 on glass plate), and com-
pounds were visualized by exposure to UV light and/or by
charring with 10% H2SO4 in ethanol. Silica gel (80 mesh and 300
mesh) manufactured by Fuji Silysia Co. was used for flash column
chromatography. Quantity of silica gel was usually estimated as
100 to 150-fold weight of sample to be charged. Reversed phase
silica gel (WakogelÒ 50C18) was purchased from Wako Pure Chem-
ical Industries, Ltd. HPLC grade water and methanol (WAKO) were
used for separation of the final compounds. Solvent systems in
chromatography were specified in v/v. All evaporations and con-
centrations were carried out in vacuo.
4.2.2. (4-Biphenyl)methyl 3,5,9-trideoxy-5-glycolamido-9-(40-
hydroxy-4-biphenyl) methylamino-
nonulopyranosidonic acid (9)
D-glycero-a-D-galacto-2-
1H NMR (CD3OD): d 7.58–7.38 (m, 11H, Ar-H), 6.85–6.83 (m, 2H,
Ar-H), 4.88 (d, J = 11.7 Hz, 1H, glycosidic CH2), 4.58 (d, J = 11.7 Hz,
1H, glycosidic CH2), 4.15 (m, 1H, H8), 4.04 (s, 2H, glycolyl CH2CO),
3.99 (s, 2H, biphCH2NH), 3.88–3.84 (m, 1H, H4), 3.79–3.72 (m, 2H,
H5, H6), 3.40 (d d, J = 8.9, 1.4 Hz, 1H, H7), 3.18 (dd, J = 4.8, 12.4 Hz,
1H, H9a), 2.94 (d d, J = 4.8, 12.4 Hz, 1H, H9b), 2.81 (dd, J = 8.9,
12.4 Hz, 1H, H3eq), 1.68 (t, J = 12.4 Hz, 1H, H3ax). 13C NMR (CD3OD):
d 177.4, 158.7, 156.6, 153.3, 143.6, 132.6, 131.6, 130.3, 129.1,
128.0, 119.3, 116.8, 115.4, 104.0, 75.3, 74.7, 74.0, 72.7, 72.4, 69.9,
69.1, 68.4, 64.1, 62.6, 56.0, 53.8, 52.0, 51.2, 42.6. MALDI-TOF MS
calcd for C37H41N2O10 (M+H)+, 673.27; found 673.28.
4.1.2. Benzyl 3,5,9-trideoxy-5-glycolamido-9-(40-fluoro-4-
biphenyl)acetamido-
D-glycero-a-D-galacto-2-
nonulopyranosidonic acid (7)
4.3. Biological assays
To the amine 3 (49.7 mg, 0.12 mmol) in water (1.5 mL) was
added solution of N-hydroxysuccinimidyl ester of 40-fluorobiphe-
nyl-4-acetic acid (52.4 mg, 0.16 mmol) in acetonitrile (5 mL), while
maintaining the pH between 8.0 and 9.0 with saturated sodium
hydrogen carbonate. The mixture was stirred at room temperature
for 48 h. The solvent was evaporated and the residue was reconsti-
tuted into water, and applied onto a silica reversed phase column
pre-equilibrated in water. The compound was eluted with a gradi-
ent of methanol–water (0:1–40:100) to afford compound 9 in
(52.6 mg, 70%) yields as a white fluffy solid after a final lyophiliza-
4.3.1. Competition ELISA
The Fc region of human IgG1 fused to the N-terminal three Ig
domains of murine and human CD22 (mCD22Fc and hCD22Fc,
respectively) was prepared from transfectants of Lec2 cells, a mu-
tant line of CHO cells deficient in protein sialylation, as described
previously.20 Binding of synthetic sialosides to CD22 was analyzed
by competition ELISA.
96-Well plates (Greiner) were coated overnight with 50
lL of
streptavidin (40 g/mL) dissolved in 50 mM NaHCO3 (pH 8 .5).
l

H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. 19 (2011) 1966–1971
1971
Plates were then incubated with 50
l
L of 4
l
g/mL of biotinylated
imposed into the BIP in the models, confirming that interactions
essential for competitive binding are maintained. Then obtained
complexes were then energetically minimized with 1000 iterations
of ‘in situ ligand minimization algorithm’ using Smart Minimizer
option using CHARMm force field. After the minimizations, molec-
ular dynamics simulations were initiated using standard dynamics
cascade module. Molecular dynamics cascade consisted of 1000
steps of steepest descent energy minimization and conjugate gra-
dient energy minimization. Then, heating of 2000 steps (initial
temperature 50 K, final temperature 300 K), equilibration of
120 ps (1 fs time step, coordinates saved every 250 steps), produc-
tion of 120 ps (1 fs time step, 300 K, NVT ensemble, nonbond cutoff
14A, switching function between 10 and 12A, coordinates saved
every 150 steps) were applied. For all the above calculations,
dielectric constants of 1 (interior) and 80 (exterior) were used.
The representative ligand binding modes were analyzed to investi-
gate the relative differences between interactions to understand
experimental selectivity differences.
form of compound 112 in PBS for 1 h. After blocking of the wells
with 0.5% BSA in PBS for 3 h, various concentrations of synthetic
sialosides were incubated for 1 h together with 0.5 lg of the
CD22Fc fusion protein. After washing with PBS containing 0.05%
Tween 20, CD22Fc bound to the plates was detected using alkaline
phosphatase-conjugated
(Southernbiotech).
anti-human
IgG
Fc
antibody
Alternatively, high-binding 96-well flat bottom plates (Nunc)
precoated with neutravidin (Pierce Biotechnology) at 1 g/50
of PBS/well overnight at 4 °C were washed with Hanks balanced
l
lL
salt solution (HBSS; 200
0.5% BSA.
lL, 3ꢁ) and blocked with HBSS containing
4.3.2. Competition ELISA (hCD22)
High-binding 96-well flat bottom microtiter plates (Nunc) pre-
coated with neutravidin (Pierce Biotechnology) at 1
PBS/well overnight at 4 °C were washed with Hanks balanced salt
solution (HBSS; 200
L, 3ꢁ) and blocked with HBSS containing
0.5% BSA for 30 min at 37 °C. Wells were then washed with HBSS
once (200 L). Fifty microliters of the Sia 2-6LacNAc-PAA-biotinyl-
ated sialoside (0.625 g/mL) was aliquoted into each well, and al-
lg/50 lL of
l
Acknowledgements
l
a
l
H.H.M.A. is gratefulto theEgyptianGovernment for Ph.D. funding
scholarship. This work was partly supported by the Ministry of Edu-
cation, Culture, Sports, Science, and Technology (MEXT) of Japan
(Grant-in-Aid for Scientific Research to M. Kiso, Nos. 1710100 and
22380007) and CRESTof JST (Japan Science and Technology Corpora-
tion) and NIH grants GM60938 and AI50143 to J. Paulson.
lowed to bind for 1 h at room temperature and free sialoside was
washed away with HBSS (200
anti-human IgG (0.6
l
L, 5ꢁ). Affinity purified F(ab0)2 goat
l
g/mL; Jackson ImmunoResearch Laborato-
ries), horseradish peroxidase (HRP), conjugated affinity purified
F(ab0)2 fragment rabbit anti-goat F(ab0)2 specific (0.3
lL/ml; Jackson
ImmunoResearch Laboratories) and hCD22-Fc21 (ꢀ1.25
lg/mL)
were mixed in PBS and allowed to precomplex for 45 min. at room
Supplementary data
temperature. 50
body complex solution were added to each well and allowed to bind
for 30 min at 37 °C before washing five times with 200 L of HBSS.
Wells were developed with 100
L of TMB solution (3,30,5,50-tetra-
lL of the inhibitor and 50 lL of the hCD22-Fc-anti-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.060.
l
l
methylbenzidine, Pierce). The reaction was stopped with 100
1 M H2SO4 and the OD was measured at 450 nm.
ll of
References and notes
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4.3.3. Competition ELISA (MAG)
As described for hCD22 but in this case fifty microliters of the
2-3LacNAc-PAA-biotinylated sialoside (0.33 M) was ali-
quoted into each well, and allowed to bind for 1 h at room temper-
ature and free sialoside was washed away with HBSS (200
L, 5ꢁ).
Affinity purified F(ab0)2 goat anti-human IgG (0.05
g/mL) and HRP
conjugated affinity purified F(ab0)2 fragment rabbit anti-goat
F(ab0)2 specific (0.0167
g/mL) were allowed to precomplex in
Sia
a
l
l
l
l
8. Kelm, S. Results Probl. Cell Differ. 2001, 33, 153.
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sialidase treated MAC-Fc21 (ꢀ0.12
lg/ml) which had been pre-
treated with V. cholerae sialidase (0.4 mU/ml) for 45 min at room
temperature), stopped by adding EDTA to 1 mM and incubated
for an additional 45 min at room temperature. The MAG-Fc-anti-
body complex solution was then diluted 3:2 with HBSS/BSA/EDTA,
50
were added to each well and allowed to bind for 30 min at 37 °C
before washing five times with 200 L of HBSS. Wells were devel-
oped with 100
L of TMB solution (3,30,5,50-tetramethylbenzidine,
lL of the inhibitor and 50 lL of the MAG-Fc-antibody complex
l
l
15. Shelke, S. V.; Gao, G. P.; Mesch, S.; Gathje, H.; Kelm, S.; Schwardt, O.; Ernst, B.
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Pierce). The reaction was stopped with 100
OD was measured at 450 nm.
ll of 1 M H2SO4 and the
4.4. Molecular modeling
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R.; Kelm, S.; Jones, E. Y. J. Mol. Biol. 2007, 365, 1469.
Manual docking and Molecular dynamics were carried out on
an Intel P4 based Microsoft windows 2000 workstation using
Discovery Studio Modeling 1.5 and 2.0 packages (Accelrys, Inc.,
San Diego, CA). The construction of the complex structure of
mCD22 with the ligand was accomplished in three steps. First,
the structures of 5 and 8 were built using BIP model complexes
as a template as reported.6 The individual antagonists were super-
19. Mesch, S.; Moser, D.; Strasser, D. S.; Kelm, A.; Cutting, B.; Rossato, G.; Vedani,
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B. J. Med. Chem. 2010, 53, 1597.
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