ˇ
K. Strancar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 343–348
348
12. Snyder, N. J.; Tebbe, M. J.; Victor, F.; Blaszczak, L. C.;
Halligan, N. G.; Sigmund, S.; Thompson, R. C.; Wilkie, S.
C.; Stack, D. R.; Lee, C.; Birch, G. B.; Wu, C. E.; Smith,
M. C. Abstracts of the 39th Interscience Conference on
Antimicrobial Agents and Chemotherapy, September 26–
29, San Francisco, USA, 1999; p 330.
13. Victor, F.; Tebbe, M. J.; Birch, G. B.; Smith, M. C.;
Letourneau, D. L.; Wu, C. E. Abstracts of the 39th
Interscience Conference on Antimicrobial Agents and Che-
motherapy, September 26–29, San Francisco, USA, 1999;
p 330.
14. Kotnik, M.; Oblak, M.; Humljan, J.; Gobec, S.; Urleb, U.;
ˇ
Solmajer, T. QSAR Comb. Sci. 2004, 23, 399.
15. Baylis, E. K.; Campbell, C. D.; Dingwall, J. G. J. Chem.
Soc., Perkin. Trans. 1 1984, 2845.
16. Bartlett, P. A.; Hanson, J. E.; Morgan, B. P.; Ellsworth, B.
A. In Houben-Weyl: Synthesis of Peptides and Peptidom-
imetics, 4th ed.; Goodman, M., Ed.; Houben-Weyl Meth-
ods in Organic Chemistry; Thieme: Stuttgart, 2003; Vol.
E22c, p 492.
a Nucleosil 5C18 column (150 · 4.6 mm) as stationary
phase, and isocratic elution at a flow rate of 0.6 ml/min
with 50 mM ammonium formate, pH 4.7. Compounds
were detected and quantified with an HPLC radioactivity
monitor LB 506 C-1 (Berthold France, Thoiry, France)
using Quickszint Flow 2 scintillator (Zinsser Analytic,
Maidenhead, UK) at 0.6 ml/min. Residual activity was
calculated with respect to
a similar assay without
inhibitor. Values are expressed as means standard
deviations of duplicate determinations. IC50 values were
determined from a range of inhibitor concentrations;
values standard deviations at 95% of confidence were
calculated from the fitted regression equations using the
logit-log plot.
18. Auger, G.; Martin, L.; Bertrand, J.; Ferrari, P.; Fanchon,
´
E.; Vaganay, S.; Petillot, Y.; van Heijenoort, J.; Blanot,
D.; Dideberg, O. Protein Expr. Purif. 1998, 13, 23.
19. All the compounds gave satisfactory spectroscopic data
and elemental analyses. Representative results for com-
pound 12c are given: mp 82–84 ꢁC; IR (KBr): m 3427, 1717,
17. Enzymatic assays were performed as described8 with
slight modifications. The compounds were tested for their
ability to inhibit the addition of D-[14C]Glu to UDP-
MurNAc-L-Ala in a mixture (final volume: 50 ll) con-
taining 0.1 M Tris–HCl, pH 8.6, 5 mM MgCl2, 25 lM
UDP-MurNAc-L-Ala, 25 lM D-[14C]Glu (50,000 cpm),
5% (v/v) DMSO, and purified MurD18 (diluted with
20 mM potassium phosphate, pH 7.0, 1 mM dithiothre-
itol and 1 mg/ml BSA) and 1 mM tested compound (all
compounds were soluble in the assay mixture containing
5% DMSO). The mixture was incubated for 30 min at
37 ꢁC, and the reaction was stopped by adding 10 ll
glacial acetic acid. The mixture was lyophilized and taken
up in the HPLC elution buffer. The radioactive substrate
and product were separated by reverse-phase HPLC with
1653, 1449, 1254, 1036, 970, 807 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 1.14–1.28 (m, 3H, –CH3), 1.53–
2.33 (m, 6H, –CH2–CH(COOH)–CH2–CH2–COOH),
2.57–2.70 (m, 1H, –CH2–CH(COOH)–CH2), 4.07–4.23
3
(m, 1H, –PCH), 6.06 (s, 2H, –O–CH2–O–), 6.59 (d, JH–
H (E) = 15.1 Hz, 1H, –HC@CH–CONH), 6.95 (d,
Jortho = 7.9 Hz, 1H, ArH-5), 7.07 (d, Jortho = 7.9 Hz, 1H,
3
ArH-6), 7.11 (s, 1H, ArH-2), 7.36 (d, JH–H (E) = 15.4 Hz,
1H, Ar-HC@CH–). 31P NMR (DMSO-d6): d 45.65 (1P),
45.27 (1P). MS (FAB): m/z 428 (M+H)+; Anal. Calcd for
C18H22NO9P · H2O: C, 48.54; H, 5.43; N, 3.14. Found: C,
48.75; H, 5.42; N, 2.99.
20. Bertrand, J. A.; Auger, J.; Martin, L.; Fanchon, E.;
Blanot, D.; Le Beller, D.; van Heijenoort, J.; Dideberg, O.
J. Mol. Biol. 1999, 289, 579.