W. Pan et al. / Tetrahedron Letters 48 (2007) 4353–4356
4355
amination between 4 and 6 or 7 required some optimiza-
tion in order to obtain satisfactory yields. Several reac-
tion parameters were screened: the solvent, the
temperature, the use of primary amine 4 as a hydrochlo-
ride salt or a free amine, the reducing agent and the
addition of weak acids such as acetic acid to promote
the imine formation. We mainly found that NaB-
H(OAc)3 gave significantly better yields than NaBH3CN
under comparable and optimized conditions, and that
the amine had to be used as its hydrochloride salt.12 De-
spite the presence of a polar secondary amine function-
ality, benzylated phosphonates 10 and 11 could easily be
purified by standard silica gel chromatography and were
isolated in 92% and 82% yield, respectively. Hydrogen-
olysis of 10 and 11 finally afforded pure target molecules
1 and 2 in quantitative yield.13
properties (Kd = 46 lM and 54% inhibition at 2.5 mM)
in the range of that of UDP (Kd = 14 lM and 51% inhi-
bition at 2.5 mM).
Therefore, we envision now to synthesize and test UDP-
aminophosphonate 3 (Scheme 1) that should display
strong inhibition properties due to the presence of a
UDP moiety, a cationic amino group and an acyclic
D-galactitol or D-lyxitol side-chain.
Work is in progress to complete the synthesis of UDP-
aminophosphonate 3 and test it as an inhibitor of
UGM.
Acknowledgment
The inhibition of the UGM catalyzed reaction by these
two final molecules was then assessed by a competition
assay: the kinetic of isomerization of UDP-Galf into
UDP-Galp was followed by HPLC in the presence and
absence of inhibitor. UGM from Escherichia coli was
overexpressed and purified as previously described.25
The inhibitions were measured under reducing condi-
tions, using freshly prepared sodium dithionite
(20 mM). The UDP-Galf and enzyme concentrations
were 150 lM and 15 nM, respectively.14
This work was supported by the University of Namur
(Post-doc grant to W.P. and Ph.D. grant to C.A.).
References and notes
1. Kremer, L.; Besra, G. S. Expert. Opin. Inv. Drug 2002, 11,
1033–1049.
2. Kremer, L.; Dover, L. G.; Morehouse, C.; Hitchin, P.;
Everett, M.; Morris, H. R.; Dell, A.; Brennan, P. J.;
McNeil, M. R.; Flaherty, C.; Duncan, K.; Besra, G. S. J.
Biol. Chem. 2001, 276, 26430–26440.
To control the assay, we also tested UDP as an inhibitor
under the same conditions. Since the Kd of UDP with
UGM has been measured, this inhibitor can now be
considered as a standard.11,15–17 At a concentration of
2 M of inhibitors we found that UDP, 1 and 2 inhibited
the reaction at 36%, 0% and 11%, respectively. In the
three cases the inhibition levels were weak, but to our
surprise D-lyxose derivative 2 reproducibly displayed
better inhibition properties than D-galactose amino-
phosphonate 1.
3. Caravano, A.; Sinay, P.; Vincent, S. P. Bioorg. Med.
¨
Chem. Lett. 2006, 16, 1123–1125.
4. Pan, F.; Jackson, M.; Ma, Y.; McNeil, M. R. J. Bacteriol.
2001, 183, 3991–3998.
5. Bornemann, S. Nat. Prod. Rep. 2002, 19, 761–772.
6. Barlow, J. N.; Blanchard, J. S. Carbohydr. Res. 2000, 328,
473–480.
7. Huang, Z.; Zhang, Q.; Liu, H.-W. Bioorg. Chem. 2003, 31,
494–502.
8. Soltero-Higgin, M.; Carlson, E. E.; Gruber, T. D.;
Kiessling, L. L. Nat. Struct. Mol. Biol. 2004, 11, 539–543.
9. Fullerton, S. W. B.; Daff, S.; Sanders, D. A. R.; Ingledew,
W. J.; Whitfield, C.; Chapman, S. K.; Naismith, J. H.
Biochemistry 2003, 42, 2104–2109.
10. Zhang, Q.; Liu, H.-W. J. Am. Chem. Soc. 2001, 123, 6756–
6766.
11. Itoh, K.; Huang, Z.; Liu, H.-W. Org. Lett. 2007, 9, 879–
882.
To date, several groups have described cationic analogs
of D-galacto furanose designed as inhibitors of either
UGM or the galactan biosynthesis. The groups of
Fleet,18,19 Thomas,20,21 and Martin22,23 described the
synthesis of iminosugars while Pinto and co-workers
described the synthesis of sulfonium and selenonium
derivatives.17 The published inhibitory activities of these
molecules are usually poor, as in the case of amino-
phosphonates 1 and 2. The structural difference of the
previously described molecules is that they are cationic
analogs of galactofuranose whereas molecules 1 and 2
are acyclic molecules (1-amino-alditols) and possess a
phosphonic acid functionality, thus mimicking a puta-
tive transient intermediate of the enzymatic reaction.
As in the case of the conformational and mechanistic
probes we published earlier,24–26 the presence of a
UDP moiety within the structure of the inhibitor seems
to be a prerequisite to ensure a tight binding with
UGM.
12. Aminophosphonate 5 (146 mg, 0.273 mmol) was sus-
pended in a solution of HCl (3 mL, 1 M) in MeOH
(3 mL). The mixture was refluxed at 50 °C for 10 min and
cooled down to room temperature. The resulting solution
was diluted with MeOH (8 mL), concentrated at 20 °C to
remove the solvents and dried under vacuum for 2 h. To
this residue was added a solution of aldehyde 6 (280 mg,
0.44 mmol) in anhydrous THF (16 mL) followed by
NaBH(OAc)3 (231 mg, 1.09 mmol) and the mixture was
stirred vigorously overnight at room temperature. Then,
the suspension was filtered through a short pad of silica gel
and the solvents were evaporated under reduced pressure.
The residue was purified by Flash Column Chromatogra-
phy (cyclohexane/EtOAc, 3:1) to afford 10 as a colorless
oil (229 mg, 92%, Rf 0.65 with cyclohexane/EtOAc 1:1).
20
13. Analytical data for 1: ½aꢀD ꢁ11.3 (c 0.9 in H2O); 1H NMR
Very recently, Liu and co-workers described the synthe-
sis of a non cationic UDP-galactitol in which the galact-
ose mimic is also acyclic.11 This substrate analog was
designed as a mechanistic probe and displayed binding
(400 MHz,
D2O):
d = 4.12
(ddd,
J2,3 = 0.9 Hz,
J2,1a = 4.3 Hz, J2,1b = 9.1 Hz, 1H; H-2), 3.79 (td,
J5,6 = 8.4 Hz, J5,4 = 0.9 Hz, 1H; H-5), 3.49 (m, 4H; H-6,
H-4 and H-3), 3.22 (ABX, J1a,1b = 13.3 Hz, J1a,2 = 4.3 Hz,
Pan, Weidong
