Deoxynojirimycin and its hexosaminyl derivatives bind to natural killer cell receptors rNKR-P1A and hCD69

Article abstract of DOI:10.1016/j.bmcl.2010.05.109

Deoxynojirimycin (1) and two new related 4-O-hexosaminyl-containing disaccharide mimics, β-d-TalNAc-(1→4)-DNJ (4) and β-d-ManNAc- (1→4)-DNJ (5), have been studied as agonists of natural killer (NK) cell receptors. As a positive and unexpected result, DNJ (1) displayed a remarkable activation effect towards both NKR-P1A (rat) and CD69 (human) receptors, and a quite similar activity was found for 4 and 5. The synthesis of the two disaccharide mimics is based on an approach that avoids the glycosylation step using known intermediates arising from lactose. The key stage of the synthesis involves the construction of the DNJ unit through an initial C-5 oxidation of the reducing d-glucopyranosyl unit followed by a stereoselective double-reductive aminocyclization of the 1,5-dicarbonyl disaccharide intermediates.

Full text of DOI:10.1016/j.bmcl.2010.05.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4645–4648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Deoxynojirimycin and its hexosaminyl derivatives bind to natural killer
cell receptors rNKR-P1A and hCD69
a
a
a
c
Giorgio Catelani a, , Felicia D’Andrea , Alessio Griselli , Lorenzo Guazzelli , Petra Nemcová ,
*
ˇ
Karel Bezouška b,c, Karel Krenek , Vladimír Kren
b,c
b,
*
ˇ
ˇ
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno, 33, I-56126 Pisa, Italy
b Institute of Microbiology, Academy of Sciences of the Czech Republic, Centre of Biocatalysis and Biotransformation, CZ 142 20 Praha 4, Czech Republic
c Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, CZ-12840 Praha 2, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Deoxynojirimycin (1) and two new related 4-O-hexosaminyl-containing disaccharide mimics, b-D-Tal-
Received 28 April 2010
Revised 27 May 2010
Accepted 29 May 2010
Available online 8 June 2010
NAc-(1?4)-DNJ (4) and b-D-ManNAc-(1?4)-DNJ (5), have been studied as agonists of natural killer
(NK) cell receptors. As a positive and unexpected result, DNJ (1) displayed a remarkable activation effect
towards both NKR-P1A (rat) and CD69 (human) receptors, and a quite similar activity was found for 4 and
5. The synthesis of the two disaccharide mimics is based on an approach that avoids the glycosylation
step using known intermediates arising from lactose. The key stage of the synthesis involves the con-
struction of the DNJ unit through an initial C-5 oxidation of the reducing D-glucopyranosyl unit followed
by a stereoselective double-reductive aminocyclization of the 1,5-dicarbonyl disaccharide intermediates.
Keywords:
Deoxynojirimycin
b-
b-
D
-TalNAc-(1?4)-DNJ
-ManNAc-(1?4)-DNJ
Ó 2010 Elsevier Ltd. All rights reserved.
D
NK cells
rNKR-P1A and hCD69 receptors
Natural killer (NK) cells are a distinct subset of lymphoid cells
that have innate immune functions, providing an early protection
against viruses, parasites, bacteria, and tumors. Recent studies
have revealed that NK cells are also involved in T-cell-mediated
adaptive immune responses via indirect mechanisms (secretion
of cytokines or chemokines, DC maturation/activation) or by direct
cell-to-cell interactions.1 Detailed studies aimed at determining
the structural requirements of optimal carbohydrate ligands for
NKR-P1, the major NK rat receptor, have established that:2 (i) the
presence of a 2-deoxy-2-acetamido group is crucial for binding;
(ii) the agonist activity is related to the relative C-2, C-4 group
orientation, determining the following activity order: Man-
NAc > GalNAc > GlcNAc ꢀ TalNAc; (iii) a group with hydrogen
bond accepting properties (–OH, –COOH, –COOR) is needed for a
strong interaction with the receptor; (iv) a b-oriented anomeric
substituent, and b-(1?4) interglycosidic connection for di- and oli-
gosaccharides are required. Recently, b-
D-TalNAc-(1?4)-D-Glc (2)
and b- -ManNAc-(1?4)- -Glc (3) disaccharides have been synthe-
D
D
sized and tested for their affinity towards natural killer cell NKR-
P1A and CD69 receptors,3 exhibiting interesting binding activities.
This prompted us to synthesize some structural analogues and/or
mimetics of 2 and 3, such as compounds 4 and 5 by replacing
OH
X
OH
X
OH
NHAc
NHAc
OH
NH
OH
O
NH.HCl
O
O
HO
O
OH
HO
O
Y
HO
HO
HO
Y
HO
.HCl
OH
OH
OH
1
2: X=OH; Y=H
3: X=H; Y=OH
4: X=OH; Y=H
5: X=H; Y=OH
* Corresponding authors. Tel.: +39 0502219678; fax: +39 0502219660 (G.C.).
E-mail addresses: giocate@farm.unipi.it, g.catelani@tin.it (G. Catelani).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.109

4646
G. Catelani et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4645–4648
the
D-glucose unit with a 1-deoxynojirimycin (DNJ, 1), and evalu-
tetraol derivative of lactose,10 in which the corresponding 40-keto
ate them as NK cell receptor agonists.
derivative was isolated as a by-product. This difference could be
attributed to a destabilization of the 30,40-stannylidene acetal ring
by the steric hindrance of the axial C-20 acetamido group. The alde-
hydo group of 8 and 9 was deprotected by treatment with 90% aq
CF3COOH, also causing the removal of the 2,3-O-isopropylidene
acetal protection. 1,5-Dicarbonyl disaccharide intermediates, com-
prised of a mixture of tautomeric forms (NMR), were submitted
without further purification to a double-reductive aminocycliza-
tion by treatment with NaBH3CN (2.2 equiv) and BnNH2ꢁHCl
(1 equiv) in MeOH at 60 °C for 12 h, followed by direct acetylation
(Ac2O/Py, 24 h) of the crude reaction mixtures. Completely pro-
tected aza-disaccharides 1013 and 1113 were isolated with accept-
able overall yields (42% and 40%, respectively). The structure and
stereochemistry of 10 and 11 were firmly established by 1H, 13C
NMR and 2D NMR experiments. In particular, diagnostic signals
for the DNJ unit were the H-1ax, H-1eq and H-5 protons located,
for example in 11, at d 2.16 (dd, J1ax,1eq 11.8 Hz, J1ax,2 10.5 Hz), at
d 2.90 (dd, J1eq,2 5.2 Hz) and at d 2.64 (dd, J4,5 9.6 Hz, J5,6a 3.1 Hz,
J5,6b 2.3 Hz). As for some reported aminocyclizations of xylo config-
ured aldohexos-5-uloses,7d,10,14 also here the intramolecular
reductive amination takes place with complete stereoselectivity,
leading to the isolation of the sole iminosugar with a gluco config-
uration, probably because of the presence of stereoelectronic and
conformational factors involving an axial hydride attack during
the stereodifferentiating step on the more stable (all equatorial)
conformer of the cyclic iminium ion intermediate.15 Finally, com-
pounds 10 and 11 were routinely deprotected via an initial de-O-
acetylation (MeONa/MeOH), followed by catalytic hydrogenolysis
(H2, Pd/C in 1.3 M methanolic HCl) leading to the target disaccha-
DNJ is by far the most investigated iminosugar, a class of carbo-
hydrate mimetics, which have been the focus of increasing atten-
tion over the last two decades because of their glycosidase
inhibitory properties.4 As a result, N-hydroxyethyl-DNJ (Glyset™)
and N-butyl-DNJ (Zavesca™) have been approved as drugs for type
II diabetes and Gaucher disease treatment, respectively. More re-
cently, DNJ and a large number of structurally related iminosugar
analogues have been synthesized and extensively studied for their
biological activities, exhibiting therapeutic potential towards a
range of diseases, including cancer, viral infections, and lysosomal
diseases.5 However, the immunomodulation properties of imino-
sugars have received little attention and their effect on the im-
mune system has only been reported in a limited number of
papers.6,7 Van den Broek et al. reported in 1996 that DNJ and N-
7-oxadecyl-DNJ inhibit the in vitro proliferation of lymphocytes.6
More recently, a contribution from a Chinese group claims that
N-pentafluorobenzyl-DNJ displays a specific inhibitory effect on
cytokine secretion from human peripheral blood mononuclear
cells (PMBC) and human CD4+ T cells.7a Further studies by the
same group reported remarkably immunosuppressant activities
of N-alkylated 1,6-dideoxy-DNJ derivatives7d and of
and
-altro-configured 1-hydroxyethyl7b and 1-acylamidoethyl7d
DNJ derivatives. Unexpectedly, the interferon- (IFN- ) secretion
D-galacto-
L
c
c
from mice splenocytes was found to be strongly enhanced by some
compounds of the latter type, thus for the first time suggesting
immunostimulating properties for specific iminosugars.7e
Several glycosylated DNJ derivatives have been isolated from
natural products exhibiting interesting biological activities.8 How-
ever, to the best of our knowledge, no DNJ derivative glycosylated
with a hexosaminyl unit has so far been isolated from natural
sources or synthesized. Taking advantage of the availability9 of
intermediates 6 and 7 from lactose, we considered an approach
for the synthesis of 4 and 5 based on the intramolecular double-
ride mimics b-
D-TalNAc- and b-D-ManNAc-(1?4)-DNJ as hydro-
chlorides (4 and 5).16
New compounds 4 and 5 were tested for binding affinity to-
wards the activation receptors of NK cells, rat NKR-P1A17, and hu-
man CD69.18 As controls for assessing the structure–activity
relationship, the respective building units were also included in
the plate inhibition assays as well as the standard positive control
GlcNAc.19 Surprisingly, DNJ gave comparable results to GlcNAc,
used in these tests as a benchmark. Coupling the two units in-
creases the binding, but with NKR-P1A it only has an additive
and not multiplicative effect. A different situation, however, was
found with CD69 in compound 5, where the attachment of
ManNAc (that is per se inactive), to DNJ (1) increases the binding
by more than one order of magnitude. Nevertheless, the most
interesting result from a practical point of view seems to be the
binding of DNJ to the activation receptors of NK cells.
reductive amination of b-D-hexosaminyl-(1?4)-D-xylo-aldohexos-
5-ulose derivatives. This approach, leading to disaccharide azami-
mics avoiding the glycosylation step of a DNJ acceptor, was pro-
posed by ourselves some years ago10 and further used by Stütz
for the synthesis of biologically relevant azadisaccharide mimics.11
The selective oxidation at C-5 of polyols 6 and 7 was performed
using the well known stannylidene acetal mediated method.12
Thus, the 5,6-O-stannylidene acetal obtained from the triol 7 by
reaction with Bu2SnO (1.0 equiv) in refluxing toluene under azeo-
tropic water removal conditions, followed by treatment with NBS
after changing the solvent from toluene to chloroform produced
the C-1 aldehydo protected ketone 913 (Scheme 1) with an excel-
lent 94% yield. A similar result was obtained for the oxidation of
the tetraol 6, leading to the corresponding ketone 813 as the sole
oxidation product isolated with a 67% yield. It is noteworthy that
also in this case the oxidation takes place with complete regiose-
lectivity, despite the possible involvement of a 30,40-O-stannylidene
acetal. This is at variance with the result reported for the analogous
In a series of previous studies,2 it was clearly demonstrated that
NKR-P1 receptor interacts strongly with N-acetylhexosamines. The
binding groove optimally binds linear oligosaccharides (typically
chitoteraose); GalNAc and ManNAc being stronger ligands than
GlcNAc. In contrast, CD69 only binds well to GlcNAc, but linked
to the branched, multiantennary structures, where multivalency
plays a crucial role.20 This is also the reason why the binding
OH
OH
OH
OBn
OBn
NHAc
OH
NHAc
OBn
NHAc
X
X
X
X
NHAc
OR'
N
OH
O
O
O
O
O
R'O
O
HO
O
Y
RO
i
ii
iii
Y
HO
Y
RO
Y
RO
O
O
.
NH HCl
Ph
O
O
OH
OR'
(MeO)2CH
(MeO)2CH
CMe2
CMe2
O
O
8: R=Y=H, X=OH
9: R=Bn, X=H, Y=OH
4: X=OH, Y=H
5: X=H, Y=OH
6: R=Y=H, X=OH
7: R=Bn, X=H, Y=OH
10: R=R'=Ac, X=OAc, Y=H
11: R=Bn, R'= Ac, X=H, Y=OAc
Scheme 1. Reagents and conditions: (i) Bu2SnO, CH3Ph, reflux, 20 h, then NBS, CHCl3, rt, 0.5–1.5 h (8: 67%; 9: 94%); (ii) (a) 90% aq TFA, rt, 2–3 h; (b) PhCH2NH2ꢁHCl, NaBH3CN,
MeOH, 60 °C, 12 h; (c) Ac2O, Py, rt, 24 h (10: 42% from 8; 11: 44% from 9); (iii) (a) MeONa, MeOH, 0 °C, 4–5 h; (b) H2, Pd/C 10%, HCl, MeOH, rt, 2.5–5 h (4: 92%; 5: 81%).

G. Catelani et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4645–4648
4647
ˇ
ˇ
Table 1
Havlícek, V.; Pospíšil, M.; Thiem, J.; Kren, V. Biochem. Biophys. Res. Commun.
1997, 238, 149.
Affinity of DNJ and its glycosyl derivatives towards two NK cell activation receptors,
NKR-P1A (rat), and CD69 (human), expressed on a logarithmic scale (ꢂlog IC50
ˇ
3. Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F.; Krenek, K.; Bezouška, K.;
)
ˇ
Kren, V. J. Carbohydr. Chem. 2008, 27, 156.
Compound
NKR-P1A
CD69
4. Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond; Stütz, A., Ed.;
Wiley-VHC: New York, 1999.
5. Iminosugars—From Synthesis to Therapeutic Applications; Compain, P., Martin, O.
R., Eds.; Wiley: Chichester, 2007.
6. Van den Broek, L. A. G. M.; Kat-van den Nieuwenhof, M. W. P.; Butters, T. D.;
van Boeckel, C. A. A. J. Pharm. Pharmacol. 1996, 48, 172.
7. (a) Zhang, X.-L.; Liu, Min.; Xie, P.; Wan, S.; Ye, J. T.; Zhou, X.; Wu, J. Bioorg. Med.
Chem. Lett. 2004, 14, 3789; (b) Ye, X.-S.; Sun, F.; Liu, M.; Li, Q.; Wang, Y.; Zhang,
G.; Zhang, L.-H.; Zhang, X.-L. J. Med. Chem. 2005, 48, 3688; (c) Zhang, L.; Sun, F.;
Li, Y.; Sun, X.; Liu, X.; Huang, Y.; Zhang, L.-H.; Ye, X.-S.; Xiao, J. ChemMedChem
2007, 2, 1594; (d) Zhou, J.; Zhang, Y.; Zhou, X.; Zhou, J.; Zhang, L.-H.; Ye, X.-S.;
Zhang, X.-L. Bioorg. Med. Chem. 2008, 16, 1605; (e) Zhang, L.; Sun, F.; Wang, Q.;
Zhou, J.; Zhang, L.-H.; Zhang, X.-L.; Ye, X.-S. ChemMedChem 2009, 4, 756.
8. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. Tetrahedron: Asymmetry 2000,
11, 1645.
9. Attolino, E.; Catelani, G.; D’Andrea, F.; Nicolardi, M. J. Carbohydr. Chem. 2004,
23, 179.
10. D’Andrea, F.; Catelani, G.; Mariani, M.; Vecchi, B. Tetrahedron Lett. 2001, 42,
1139.
DNJꢁHCl (1)
TalNAc-DNJꢁHCl (4)
ManNAc-DNJꢁHCl (5)
TalNAc
ManNAc
GlcNAc
5.0
5.5
6.5
4.6
6.7
5.6
3.2
3.3
4.5
0
0
3.5
Data are presented as average values from three independent experiments.
affinities with simple oligosaccharidic ligands (see Table 1) are
much weaker in CD69, where the presence of multivalent ligands
improves the binding by several orders of magnitude. Typically,
binding affinity correlates well with the killing activity of the
respective NK cells towards tumor or virally infected somatic cells.
In summary, probably the most interesting result is the bind-
ing of DNJ itself to NK cell activation receptors. DNJ is an imino-
11. (a) Steiner, A. J.; Stütz, A. E. Carbohydr. Res. 2004, 339, 2615; (b) Spreitz, J.;
Stütz, A. E. Carbohydr. Res. 2004, 339, 1923; (c) Steiner, A. J.; Schitter, G.; Stütz,
A. E.; Wrodnigg, T. M.; Tarling, C. A.; Whithers, S. G.; Fantur, K.; Mahuran, D.;
Pashke, E.; Tropak, M. Bioorg. Med. Chem. 2008, 16, 10216.
12. David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.
13. All new compounds were fully characterized with NMR and gave correct
sugar active as a selective
a-glucosidase inhibitor and is used for
the treatment of various viral infections—typically HIV infections.
During such treatment, the level of activity of the immune system
is of utmost importance and compromising it can lead to the fail-
ure of the treatment. The immunomodulatory activity of these
iminosugars has rarely been studied and only a few reports can
be found in the literature, mostly describing the immunosuppres-
sive activity of such compounds.6,7 Van den Broeck6 tested a ser-
ies of DNJ-N-alkyl derivatives and found that an N-7-octadecyl
derivative inhibited PBMC (lymphocyte)-induced proliferation.
Unfortunately DNJ was not tested. Ye et al.7b tested a series of
newly prepared iminosugar derivatives for their effects on the
elemental analysis. 8: syrup, [
MeOH); 9: syrup, [
ꢂ65.4 (c 1.12, CHCl3), Rf 0.25 (95:5 CHCl3–MeOH); 10:
solid foam, [
ꢂ29.4 (c 1.14, CHCl3), Rf 0.40 (EtOAc); 11: solid foam, [
ꢂ46.4 (c 1.1, CHCl3), Rf 0.24 (4:6 hexane–EtOAc); 4: solid foam, [
1.0, MeOH); 5: solid foam, [
a]
D
ꢂ64.6 (c 1.06, CHCl3), Rf 0.41 (8:2 CHCl3–
a
]
D
a
]
a]
D
ꢂ17.2 (c
D
a
]
D
a
]
D
ꢂ21.8 (c 1.04, MeOH).
14. Matos, C. R. R.; Lopes, R. S. C.; Lopes, S. C. Synthesis 1999, 4, 571.
15. Stevens, R. V. Acc. Chem. Res. 1984, 17, 289.
16. Compound 4: 1H NMR (CD3OD–D2O, 600 MHz): d 4.72 (br s, 1H, H-10), 4.40 (br
d, 1H, J2 ,3 3.9 Hz, H-20), 3.96 (dd, 1H, J5,6a 4.4 Hz, J6a,6b 12.2 Hz, H-6a), 3.85–
3.80 (m, 3H, H-2, H-6b, H-60b), 3.79–3.70 (m, 4H, H-4, H-60a, H-30, H-40), 3.57
(m, 1H, H-50), 3.54 (t, 1H, J2,3 = J3,4 8.5 Hz, H-3), 3.32 (dd, 1H, J1eq,2 4.9 Hz, J1ax,1eq
11.7 Hz, H-1eq), 3.21 (m, 1H, H-5), 2.89 (t, 1H, J1ax,2 11.7 Hz, H-1ax), 1.98 (s, 3H,
0
0
13
MeCON); C NMR (CD3OD–D2O, 62.9 MHz): d 174.4 (C@O), 102.1 (C-10), 79.0
secretion of IL-4 and IFN-
c from mouse splenocytes and found
(C-4), 78.0 (C-50), 76.1 (C-3), 69.5 (C-2), 68.9, 68.4 (C-30, C-40), 62.6 (C-60), 60.3
(C-5), 58.3 (C-6), 53.9 (C-20), 46.8 (C-1), 23.4 (MeCON). Compound 5: 1H NMR
that some compounds are strong immunosuppressants with po-
tential for use in the treatment of autoimmune diseases. Recently,
Zhou et al.7d prepared a series of 1,6-dideoxy-N-alkyl iminosugars
(CD3OD–D2O, 600 MHz): d 4.82 (br s, 1H, H-10), 4.55 (br d, 1H, J2 ,3 4.1 Hz, H-
20), 3.89 (dd, 1H, J5,6b 2.1 Hz, J6a,6b 11.8 Hz, H-6b), 3.87 (dd, 1H, J5,6a 4.1 Hz, H-
6a), 3.80 (m, H, H-60a, H-60b), 3.77 (dd, 1H, J3,4 8.9 Hz, J4,5 9.1 Hz, H-4), 3.73
0
0
and demonstrated IFN-
c and IL-4 inhibition activity in some of
0
0
(ddd, 1H, J1eq,2 4.9 Hz, J1ax,2 11.7 Hz, J2,3 8.9 Hz, H-2), 3.65 (dd, 1H, J2 ,3 4.1 Hz,
J3 ,4 9.6 Hz, H-30), 3.52 (t, 1H, J3,4 8.9 Hz, H-3), 3.47 (t, 1H, J4 ,5 9.6 Hz, H-40),
3.31 (m, 1H, H-50), 3.29 (dd, 1H, J1ax,1eq 11.7 Hz, H-1eq), 3.21 (m, 1H, H-5), 2.88
(t, 1H, H-1ax), 2.01 (s, 3H, MeCON); 13C NMR (CD3OD–D2O, 62.9 MHz): d 176.0
(C@O), 101.1 (C-10), 78.7 (C-50), 78.4 (C-4), 76.2 (C-3), 73.9 (C-30), 68.4 (C-2),
68.2 C-40), 61.9 (C-60), 60.3 (C-5), 58.3 (C-6), 54.5 (C-20), 47.0 (C-1), 22.8
(MeCON).
0
0
0
0
them. None of the above studies tested non-derivatized DNJ as
a control (benchmark) (1), which we consider a drawback.
Here we are reporting new results on the potential immunoac-
tivation effect of DNJ (1) which could be of significant importance
for the treatment of viral infections, during which the activation of
NK cells represents one of the critical elements of the immune re-
sponse.21,22 This pilot study indicates that DNJ and mainly its
derivatives currently used in therapy should be further tested for
their immunostimulating activities in more complex cellular sys-
tems, and in vivo.
17. For the preparation of soluble rNKR-P1A, an optimized construct was
employed involving amino acids A90 to K215 in the extracellular ligand
binding domain. After expression in bacteria, the protein precipitated into
inclusion bodies, which were dissolved in 6 M guanidine chloride in pH 9.0,
50 mM Tris–HCl and 10 mM DTT, and denatured by rapid dilution into a 100-
fold excess of refolding buffer composed of pH 9.0, 50 mM Tris–HCl
containing 1 M
L-arginine, 100 mM CaCl2, 9 mM cysteamine, 3 mM
cystamine, and 1 mM NaN3. The refolding mixture was dialyzed against a
low salt buffer composed of 15 mM Tris–HCl pH 9.0 with 9 mM NaCl and
1 mM NaN3. The diluted protein was recovered by anion exchange
Acknowledgments
chromatography on Q-Sepharose FF, and eluted using
a gradient elution
increasing the concentration of NaCl from 20 mM to 1 M. The protein, eluted
as a symmetrical peak at 0.2 M NaCl, was concentrated and further purified
by gel filtration in a Superdex 200 HR column followed by a second anion
exchange chromatography stage in a SOURCE 15Q 4.6/100 PE column. The
final preparation was homogenous on SDS PAGE, displaying a significantly
higher mobility under nonreducing conditions, and ESI-FT-ICR mass
spectrometry producing homogenous protein species at [M+H]+ = 14233.99.
The thermal and long-term biochemical stability of the protein was
investigated using spectral techniques and mass spectrometry, and the
protein proved stable for at least 7 days at 30 C or 37 °C.
This work was supported by Ministero dell’Istruzione, dell’Uni-
versità e della Ricerca (MIUR Rome, Italy, COFIN-2006 national
programme), by ESF COST Chemistry Action D34, Ministry of Edu-
cation of the Czech Republic projects OC 136, MSM21620808, and
LC06010, and by Research Concept of the Institute of Microbiology
(AVOZ50200510).
Thanks are also due to Tiziana Gragnani who contributed to the
synthesis of compound 4 within the framework of her undergrad-
uate thesis.
ˇ
ˇ
18. Vanek, O.; Nálezková, M.; Kavan, D.; Borovicková, I.; Pompach, P.; Novák, P.;
ˇ
´
Kumar, V.; Vannucci, L.; Hudecek, J.; Hofbauerová, K.; Brynda, J.; Kopecky, V.,
ˇ
ˇ
ˇ
Jr.; Kolenko, P.; Dohnálek, J.; Kaderávek, P.; Chmelík, J.; Gorcík, L.; Zídek, L.;
ˇ
Sklenár, V.; Bezouška, K. FEBS J. 2008, 275, 5589.
References and notes
19. For plate binding and inhibition assays, soluble rNKR-P1 and hCD69 proteins
were labeled by the covalent attachment of fluorescent labels through
reaction with N-hydroxysuccinimide fluorescein and rhodamine (Pierce,
Rockford, IL, USA), respectively. Four moles of fluorescein and five moles of
rhodamine were attached per mole protein as confirmed by quantitative
spectrophotometry, MALDI-MS, and ion cyclotron FT-MS. Flexible 96-well
round-bottomed polyvinyl chloride plates (BD Biosciences) were coated with
1. Santoni, A.; Zingoni, A.; Cerboni, C.; Gismondi, A. Am. J. Reprod. Immunol. 2007,
58, 280.
2. (a) Krist, P.; Herkommerová-Rajnochová, E.; Rauvolfová, J.; Semenuk, T.;
ˇ
ˇ
ˇ
Vavrušková, P.; Pavlícek, J.; Bezouška, K.; Petruš, L.; Kren, V. Biochem. Biophys.
ˇ
ˇ
Res. Commun. 2001, 287, 11; (b) Bezouška, K.; Sklenár, J.; Dvoráková, J.;

4648
G. Catelani et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4645–4648
GlcNAc17BSA ligand, blocked with 2% BSA, and then incubated in the presence
of fluorescein-rNKR-P1A or rhodamine-hCD69 (0.1 g/ml, corresponding to
half-saturation under the given experimental conditions) and serial dilutions
of the inhibitor. After incubation at 4 °C for 2 h, plates were washed four
times with PBS, and drained. The ligand-bound receptor was dissociated by
and the IC50 values were calculated from at least three independent
experiments.
l
ˇ
ˇ
20. (a) Krenek, K.; Kuldová, M.; Hulíková, K.; Stibor, I.; Lhoták, P.; Dudic, M.; Budka,
ˇ
J.; Pelantová, H.; Bezouška, K.; Fišerová, A.; Kren, V. Carbohydr. Res. 2007, 342,
ˇ
1781; (b) Bezouška, K.; Kren, V.; Kieburg, C.; Lindhorst, T. K. FEBS Lett. 1998,
ˇ
´
overnight incubation in 100
l
l of 0.1 M sodium acetate buffer supplemented
426, 243; (c) Pavlícek, J.; Sopko, B.; Ettrich, R.; Kopecky, V.; Baumruk, V.; Novák,
ˇ
´
ˇ
with 0.1% octyl-b-glucoside and 0.1% Triton X-100, the solution transferred to
96-well flat-bottomed UV transparent plates (UV Star, BioOne, Greiner), and
the amount of bound protein determined by fluorescence using standard
fluorescein and rhodamine settings for a Safire2 spectrofluorometer (Tecan,
AT). Complete inhibition curves were constructed using SigmaPlot software,
P.; Havlícková, K.; Vrbacky, M.; Martínková, L.; Kren, V.; Pospíšil, M.; Bezouška,
K. Biochemistry 2003, 42, 9295.
21. Andoniou, C. E.; Andrews, D. M.; Degli-Esposti, M. A. Immunol. Rev. 2006, 214,
239.
22. Van Dommelen, S. L.; Degli-Esposti, M. A. Immunol. Cell Biol. 2004, 82, 332.