Dowlut et al.
and strong binding affinities10,11 (with a range of 107-
108 M-1) to saccharides. For instance, PNA lectin12 binds
to the T-antigen with Ka ) 1 × 107 M-1, while Con A
lectin11 has a strong binding affinity to glycopeptides
containing 3,6-di-O-R-D-mannopyranosyl-R-D-mannopy-
ranoside (Ka ) 2.4 × 107 M-1). It should be kept in mind,
however, that these binding constants are for soluble
oligosaccharides and that the binding constants of the
same oligosaccharide structures linked to agarose beads
may be substantially different due to both the linking
arms used and to possible steric interference from the
solid support. The solid support chosen for this study is
beaded agarose, which is compatible with water, the ideal
biologically relevant solvent for reactions involving depro-
tected carbohydrates. Thus, results with agarose can
serve as a useful guide for other water-compatible
supports such as Tentagel resins or aqueous biphasic
assays such as ELISA.
Therefore, on the basis of the values of binding
constants found in the literature, the chosen ligand for
PNA lectin would be 2-aminoethyl (â-D-galactopyranosyl)-
(1f3)-2-acetamido-2-deoxy-R-D-galactopyranoside (5) hav-
ing the core structure of the T-antigen,13 with an ami-
noethyl group to attach the fluorescent label. 2-Aminoethyl
3,6-di-O-R-D-mannopyranosyl-R-D-mannopyranoside (10)
is the ligand of choice for Con A.
The ligand for the agarose-PNA conjugate was syn-
thesized as follows (Scheme 1). The acetimidate groups
were introduced at the anomeric and 3 positions of the
glycoside by reaction with DBU and trichloroacetoni-
trile.14,15 Glycosylation with 2-azidoethanol16 under Lewis
acid catalysis gave only the R anomer. This unusual and
unexpected R-selectivity was discovered while developing
a facile synthesis of R-GalNAc-serine15 and was at-
tributed to intramolecular neighboring group participa-
tion of the benzylidene oxygen atoms, which would
prevent attack by the alcohol from the â face. The
reaction is very sensitive to the protecting-group pat-
terns, but it works well in this case. This operation was
followed by the removal of the acetimidate group, in a
subsequent step, to yield the donor 2, which underwent
a glycosylation reaction17 with tetra-O-acetyl galactopy-
ranosyl bromide 3 in a nitromethane/toluene mixture (1:
1) at 70 °C, affording 4 with a yield of 61%. After removal
of the benzylidene group and acetate groups, the final
disaccharide product 5 was obtained.
FIGURE 1. (a) Favorable interactions between the agarose-
supported PNA lectin and labeled disaccharide. (b) Unfavor-
able “nonspecific” interactions between the agarose-supported
PNA lectin and labeled disaccharides 5a-c.
Unfortunately, most organic dyes contain several
aromatic rings that can induce weak “nonspecific” inter-
actions with the aromatic units of the protein receptor.
Consequently, positive hits are hard to distinguish in a
definite way from negative ones, especially at high
concentrations of the fluorescent species.
To avoid this type of nonspecific interaction, a suitable
labeling reagent would be a small molecule with a
minimum of intrinsic charges. Such a labeling reagent
would also eliminate the need for using unusual assay
conditions such as high salt and detergent-containing
buffers that help reduce problems of nonspecificity. Upon
inspecting potential new dyes recently developed for
noncarbohydrate ligands, we anticipated that succinim-
idyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hex-
anoate (NBD-X) could be an ideal labeling reagent.
Unfortunately, studies comparing labeling dyes are rare.
Herein, we describe a semiquantitative study comparing
NBD-labeled carbohydrates7 with carbohydrates labeled
with other commonly used dyes.8 This model study
(Figures 1 and 2) can be of use not only to the field of
carbohydrate chemistry but also in the screening of other
molecules.
We then proceeded to the synthesis of ligand 10 for
Con A lectin (Scheme 2). The peracetylated mannose was
converted into the bromide to serve as the acceptor in
(9) The abbreviations used are: PNA, peanut agglutinin; Con A,
concanavalin A; GlcNAc, N-acetylglucosamine; PBS, saline phosphate
buffer.
Results and Discussion
(10) It was reported by Baenziger’s group that glycopeptides with
association constants in the range of (4.5-25) × 106 M-1 are retained
by Concanavalin A-sepharose and those with association constants in
the range of (0.3-4.0) × 106 M-1 are not retained.
(11) Baenziger, J. U.; Fiete, D. J. Biol. Chem, 1979, 254, 2400-
2407.
Design and Synthesis. Our biphasic screening model
consisted of two different lectins immobilized on agarose
beads, namely PNA lectin (Arachis hypogoea) and Con
A lectin (Canavalia ensiformis).9 These lectins were
chosen because of their commercial availability, low cost,
(12) Neurohr, K. J.; Young, N. M.; Mantsch, H. H. J. Biol. Chem.
1980, 255, 9205-9209.
(7) (a) Chen, J.; Profit, A. A.; Prestwich, G. D. J. Org. Chem. 1996,
61, 6305-6312. (b) Honda, S.; Okeda, J.; Iwanaga, H.; Kawakami, S.;
Taga, A.; Suzuki, S.; Imai, K. Anal. Biochem. 2000, 286, 99-111. (c)
Sugimoto T.; Wada, Y.; Yamamura, S.; Ueda, M. Tetrahedron 2001,
57, 9817-9825. (d) Suzuki, S.; Honda, S. Electrophoresis 2003, 24,
3577-3582.
(13) T-Antigen is the disaccharide â-Gal-(1-3)-R-D-Gal-NAc, which
is O-linked to serine and threonine.
(14) Yule, J. E.; Wong, T. C.; Gandhi, S. S.; Qiu, D.; Riopel, M. A.;
Koganty, R. R. Tetrahedron Lett. 1995, 36, 6839-6842.
(15) Qiu, D.; Koganty, R. R. Tetrahedron Lett. 1997, 38, 961-
964.
(8) (a) Zhang, Y.; Le X., Dovichi, N. J.; Compston, C. A.; Palcic, M.
M.; Diedrich, P.; Hindsgaul, O. Anal. Biochem. 1995, 227, 368-376.
(b) Stoll, M. S.; Feizi, T.; Loveless, R. W.; Chai, W.; Lawson, A. M.;
Yuen, C. T. Eur. J. Biochem. 2000, 267, 1795-1804. (c) Drummond,
K. J.; Yates, E. A.; Turnbull, J. E. Proteomics 2001, 1, 304-310. (d)
Arnosti, C. J. Chromatogr. B 2003, 793, 181-191.
(16) (a) Chernyak, A. Y.; Sharma, G. V. M.; Kononov, L. O.; Krishna,
P. R.; Levinsky, A. B.; Kochetkov, N. K. Carbohydr. Res. 1992, 223,
303-309. (b) Forster, M. O.; Fierz, H. E. J. Chem. Soc. 1908, 93, 1174-
1179.
(17) Ratcliffe, R. M.; Baker, D. A.; Lemieux, R. U. Carbohydr. Res.
1981, 93, 35-41.
9810 J. Org. Chem., Vol. 70, No. 24, 2005