relatively modest.5 Inhibitors that incorporate the uridine
portion of the substrate bind substantially better, with
affinities that approximate that of UDP-Galp (Kd ) ∼50
µM).6,7 Recently, we and others have identified several non
substrate-based molecules as ligands.4,8-10 These compounds
bind much more tightly (Kd ) 2-3 µM). Moreover, we
showed that UGM inhibitors can block mycobacterial growth.
Insight into the factors that lead to effective UGM ligands
could guide the development of yet more potent inhibitors.
Our efforts to generate high-affinity UGM ligands were
informed by our previous design of a fluorescent UGM
ligand.9 We concluded that the UDP moiety of the substrate
contributes the majority of the binding energy,9 and subse-
quent studies provide additional support.6,11 Accordingly, we
tethered a fluorophore to UDP through the diphosphoryl
group. UDP binds to the UGM from K. pneumoniae
(UGMkleb) with an affinity of 26 µM and to the homologue
from M. tuberculosis (UGMmyco) with an affinity of 15 µM;
therefore, we expected the UDP-fluorescein probe to bind
with an affinity in the same range. In contrast, the probe is
a potent ligand, with an affinity approximately 100-fold
higher than that of UDP. Specifically, its Kd for UGMkleb is
0.10 µM and that for UGMmyco is 0.16 µM. The finding that
the addition of a fluorescein group enhances affinity suggests
that the fluorophore can access a secondary binding site on
the enzyme.
phosphotriester.12 Hydrogenolysis of the benzyl groups
afforded the phosphates 1a-1e as the triethylamine salts,
and these were coupled to uridine 5′-monophosphate (UMP)-
N-methylimidazolide.13 The trifluoroacetamide group was
removed and UDP derivatives 2a-2e were treated with
fluorescein isothiocyanate (FITC) to yield conjugates 3a-3e.
The affinities of the UDP-fluorescein conjugates for
UGMkleb and UGMmyco were determined using fluorescence
polarization (Table 1, Figure 2).9 The dissociation constants
Table 1. Dissociation Constants for the Complexes of UGM and
UDP-Fluorescein Conjugates 3a-3e (Scheme 1)
compound
UGMkleb Kd (µM)
UGMmyco Kd (µM)
UDP
3a
3b
26
>30
1.9
15
>30
2.5
3c
3d
3e
0.19
0.045
0.070
0.17
0.054
0.064
If the fluorescein moiety occupies an adjacent subsite, the
linker separating it and the UDP moiety should influence
binding. To test this hypothesis, we synthesized a panel of
UDP-fluorescein derivatives in which the linker was varied
systematically. We tethered the fluorophore to the nucleotide
using alkyl linkers composed of two, four, six, eight, and
ten methylene units (Scheme 1). The linker units were
Scheme 1. Synthesis of UDP-Fluorescein Conjugates
Figure 2. Relative affinity of conjugates 3a-3e for UGM. The
affinities are shown relative to that of UDP (1). An increase is
observed as the alkyl linker separating the UDP and fluorescein
moieties is extended.
of derivatives 3a-3e for either UGMkleb or UGMmyco vary
with linker length. Compound 3a, with the short two
methylene linker, binds poorly (Kd > 30 µM); the next
compound in the series, 3b, is slightly more potent (5- to
10-fold) than UDP. Conjugate 3c, which was used in our
initial fluorescence polarization-based screen, binds about
100-fold better. The compound with the eight-methylene
(5) (a) Lee, R. E.; Smith, M. D.; Nash, R. J.; Griffiths, R. C.; McNeil,
M.; Grewal, R. K.; Yan, W. X.; Besra, G. S.; Brennan, P. J.; Fleet, G. W. J.
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Sanders, D. A. R.; Pinto, B. M. Carbohydr. Res. 2004, 339, 2205–2217.
(6) Itoh, K.; Huang, Z. S.; Liu, H. W. Org. Lett. 2007, 9, 879–882.
(7) (a) Caravano, A.; Vincent, S. P.; Sinay¨, P. Chem. Commun. 2004,
1216–1217. (b) Caravano, A.; Mengin-Lecreuix, D.; Brondello, J. M.;
Vincent, S. P.; Sinay¨, P. Chem.-Eur. J. 2003, 9, 5888–5898. (c) Pan, W. D.;
Ansiaux, C.; Vincent, S. P. Tetrahedron Lett. 2007, 48, 4353–4356. (d)
Scherman, M. S.; Winans, K. A.; Stern, R. J.; Jones, V.; Bertozzi, C. R.;
McNeil, M. R. Antimicrob. Agents Chemother. 2003, 47, 378–382.
assembled from commercially available amino alcohol build-
ing blocks. Each amino alcohol was first protected as a
trifluoroacetamide12 and then converted into a dibenzyl
194
Org. Lett., Vol. 11, No. 1, 2009