gave the target molecule 4 and the a-isomer, which were readily
separated by column chromatography.
Coupled MraY–MurG radiochemical assay
12.5 ll of freshly solubilised MraY was added directly to unde-
caprenyl phosphate (0.25 lg). 12.5 ll of buffer (400 mM Tris-HCl
pH 7.5, 100 mM MgCl2) was added followed by 9 ll of water, 5 ll
of UDP-MurNAc-pentapeptide solution (1 mM), 1 ll of MurG
solution (100 lg protein ml−1) and 5 ll of DMSO or inhibitor
solution (127 mM in DMSO). The mixture was incubated at
35 ◦C for 15 minutes and then 5 ll of UDP-[3H]GlcNAc (10 lM,
500 mCi mmol−1) was added and the mixture was incubated for a
further 15 minutes. The reaction was stopped by the addition of
50 ll of pyridinium acetate pH 4.6. 100 ll of n-butanol and 100 ll
of water were then added and the layers were mixed and then
separated by centrifugation. 100 ll of the top n-butanol phase
was removed, 50 ll of fresh n-butanol was added to it, and it
was then extracted with 100 ll of water. 100 ll of the n-butanol
phase was then removed and analyzed for radioactivity. Typically,
this procedure yielded 1000–2000 cpm per assay; duplicate assays
were routinely carried out, and yielded consistent data ( 10%).
Control incubations were carried out containing no inhibitor, no
enzyme, and 50 lM ramoplanin (MurG inhibitor). Micrococcus
flavus membranes, UDP-MurNAc-pentapeptide and Escherichia
coli MurG were all prepared as described previously.23–25
The stereochemistries of products 3 and 4 were established by
2D NMR NOESY experiment of compounds 11 (the precursor of
compound 3) and 4, respectively. As shown in Fig. 2, correlations
between H1ꢀꢀ (5.15 ppm) and H4ꢀꢀ (4.08–4.13 ppm) were clearly
observed in 4, which was identified as having the b-configuration at
the C-1ꢀꢀ position. As for compound 11, the chemical shifts of H4ꢀꢀ,
H3ꢀꢀ and H5ꢀb were overlapped, so the correlations between H1ꢀꢀ
and H4ꢀꢀ were not easily identified. However, correlations between
H1ꢀꢀ (4.93 ppm) and H4ꢀ (3.68 ppm) were clearly observed in 11,
which could thus be identified as having the b-configuration at the
C-1ꢀꢀ position.
Fig. 2 NOE correlation of compounds 4 and 11.
Compounds 3 and 4 were evaluated for activity as an inhibitor of
MraY. A coupled MraY–MurG radiochemical assay was utilized,
whereby Micrococcus flavus membranes containing high levels of
MraY23 were solubilised and used to generate lipid intermediate
I in situ, to which was added purified Escherichia coli MurG and
Acknowledgements
The National Natural Science Foundation of China, the Ministry
of Education of China and Shanghai Municipal Scientific Com-
mittee are greatly acknowledged for funding this work.
3
UDP-[3H]GlcNAc. The H-labelled lipid intermediate II was ex-
tracted into n-butanol and analyzed for radioactivity. Compound
3 demonstrated 29% inhibition of MraY at a concentration of
11.4 mM, whereas compound 4 showed no inhibition at 10 mM
concentration.
References
1 W. G. Struve, R. K. Sinha and F. C. Neuhaus, Biochemistry, 1966, 5,
82.
2 D. S. Boyle and W. D. Donachie, J. Bacteriol., 1998, 180, 6429.
3 L. A. Mitscher, S. P. Pillai, E. J. Gentry and D. M. Shankel, Med. Res.
Rev., 1999, 19, 477.
4 K. Isono, M. Uramoto, H. Kusakabe, K. Kimura, K. Izaki, C. C.
Nelson and J. A. McCloskey, J. Antibiot., 1985, 38, 1617.
5 K. Kimura, Y. Ikeda, S. Kagami, M. Yoshihama, K. Suzuki, H. Osada
and K. Isono, J. Antibiot., 1998, 51, 1099.
6 C. Dini, P. Collette, N. Drochon, J. C. Guillot, G. Lemoine, P. Mauvais
and J. Aszodi, Bioorg. Med. Chem. Lett., 2000, 10, 1839.
7 A. Takatsuki, K. Kawamura, M. Okina, Y. Kodama, T. Ito and G.
Tamura, Agric. Biol. Chem., 1977, 41, 2307.
8 (a) C. Dini, N. Drochon, S. Feteanu, J. C. Guillot, C. Peixoto and
J. Aszodi, Bioorg. Med. Chem. Lett., 2001, 11, 529; (b) C. Dini, N.
Drochon, J. C. Guillot, P. Mauvais, P. Walter and J. Aszodi, Bioorg.
Med. Chem. Lett., 2001, 11, 533; (c) C. Dini, S. Didier-Laurent, N.
Drochon, S. Feteanu, J. C. Guillot, F. Monti, E. Uridat, J. Zhang and
J. Aszodi, Bioorg. Med. Chem. Lett., 2002, 12, 1209.
9 (a) Organofluorine Chemistry: Principles and Commerical Applications,
ed. R. E. Banks, B. E. Smart and J. C. Tatlow, Plenum, New York,
1994; (b) J. T. Welch, S. Eswaraksrishnan, Fluorine in Bioorganic
Chemistry, Wiley, New York, 1991; (c) Biomedical Frontiers of Fluorine
Chemistry (ACS Symposium Series 639), ed. I. Ojima, J. R. McCarthy
and J. T. Welch, American Chemical Society, Washington, DC, 1996;
(d) Organofluorine Chemicals and Their Industrial Applications, ed.
R. E. Banks, Ellis Harwood, New York, 1979; (e) R. Peters, Carbon-
Fluorine Compounds: Chemistry, Biochemistry and Biological Activities.
A Ciba Foundation Symposium, Elsevier, Amsterdam, 1972.
10 L. Pauling, The Nature of the Chemical Bond, 3rd edn, Cornell
University Press, Ithaca, NY, 1960, p. 93.
In summary, two gem-difluoromethylenated nucleoside ana-
logues of liposidomycins, 3 and 4, were both synthesized. Com-
pound 3 was assembled from lactol 5 and gem-difluoromethyl-
enated nucleoside 6. The neighbouring group participation of the
2-O-acetyl group in 5 ensured the construction of the 1,2-trans-
b-furanoside linkage during the glycosylation reaction, which
resulted in the stereocontrolled formation of 3. In assembling 4, the
trichloroacetimidate derivative of the gem-difluoromethylenated
lactol 7 was coupled with nucleoside 9 with TMSOTf as an acti-
vator in CH3CN. However, acetonitrile was found to participate
as a nucleophile in the glycosylation reaction, resulting in the
production of 16. To avoid this undesirable reaction, nitromethane
was used as the solvent, resulting in the desired product 17,
from which the target molecule 4 was prepared in a few steps.
Compound 3 showed low activity as an inhibitor of MraY.
Experimental
Solubilisation of MraY
100 ll of Micrococcus flavus membranes23 (19 mg protein ml−1)
was added to 150 ll of solubilisation buffer (50 mM Tris-HCl
pH 7.5, 1 mM MgCl2, 2 mM 2-mercaptoethanol). The mixture was
shaken at 4 ◦C for 30 minutes and then centrifuged at 13 000 rpm
for 30 minutes. The supernatant had a protein concentration of
1.5 mg ml−1 and was used directly in the radiochemical assay.
11 (a) L. W. Hertel, J. S. Kroin, J. W. Missner and J. M. Tustin, J. Org.
Chem., 1988, 52, 2406; (b) W. Plunkett, V. Gandhi, C. Chubb, B. Nowak,
V. Heinemann, S. Mineishi, A. Sen, L. W. Hertel and G. B. Grindey,
Nucleosides Nucleotides, 1989, 8, 775; (c) V. W. T. Ruiz, V. Haperen,
160 | Org. Biomol. Chem., 2008, 6, 157–161
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