DFPA can mimic 2-OG, triggering the formation of
nitrogen-fixing heterocysts and inducing a series of cellular
responses by readjusting metabolism in Anabaena.5 Using
another nonmetabolizable probe, 2-MPA (2-methylene-
pentanoic acid, Scheme 1), which employs a vinyl group to
mimic the ketone carbonyl function in 2-OG, we were able
to confirm that it is the ketone form and not the ketal form
of 2-OG that plays a signaling role during nitrogen starva-
tion in Anabaena.6 Subsequent structure/activity relation-
ship analysis with different analogs bearing various
functional groups to mimic the carbonyl group in 2-OG
underlined the importance of this group in its signaling
function.6 Only closely resembling structural motifs such
as difluoromethylene and vinyl moieties could replace the
carbonyl group to ensure the signaling function of 2-OG in
Anabaena, with DFPA and 2-MPA being the best
examples.6
Spurred on by curiosity, we then developed a hybrid
probe based on DFPA and 2-MPA, namely, DFMPA (2-
difluoromethylene-4-methylenepentanoic acid, Scheme 1)
for the structure/activity relationship study. DFMPA
contains both difluoromethylene and vinyl groups at the
C2 and C4 positions, respectively. The dual presence of
these two structural motifs would appear to considerably
change the structure of DFMPA compared to 2-OG.
Consequently, it was expected that DFMPA would neither
resemble 2-OG nor mimic its signaling role. However,
surprisingly DFMPA could mimic the signaling function
of 2-OG to induce heterocysts in Anabaena, in a way
similar to both DFPA and 2-MPA, thus suggesting that
structural alteration may be tolerated for 2-OG to exercise
its signaling role. In order to understand this, computer
modeling was carried out to investigate the interaction
between DFMPA and NtcA, the 2-OG receptor which
senses thenitrogen starvation statusvia changes in the level
of 2-OG in Anabaena.5ꢀ8 Interestingly, DFMPA was
favorably accommodated in the binding site of NtcA via
mutual conformational adaptation, further confirming the
ability of DMFPA to mimc 2-OG and execute its signaling
role. Here, we present our data supporting DFMPA’s
ability to mimic 2-OG signaling in Anabaena and provide
our rationale for the structural alteration on the basis of
computer modeling.
Scheme 2. Synthesis of DFMPA
compositionofDFMPA. However, due to crystal packing,
the crystal structures in the solid state may differ from the
corresponding structures in solution.
We next studied the ability of DFMPA to mimic the
2-OG signaling function in Anabaena. This cyanobacter-
ium is an excellent model to investigate the signaling role of
2-OG in nitrogen metabolism, since it produces morpho-
logically distinct heterocysts in response to combined-
nitrogen deprivation.10 These heterocysts are able to fix
nitrogen from air and hence allow Anabaena to survive
even under nitrogen depletion.11 Importantly, since het-
erocysts differ morphologically from vegetative cells, they
can be easily observed with light microscopy. Further-
more, heterocyst differentiation can be repressed when a
combined nitrogen source such as ammonium or nitrate is
present in the growth medium.
However, DFMPA cannot be taken up efficiently by
Anabaena since it is negatively charged at physiological
conditions.12 We therefore used a recombinant strain of
Anabaena expressing a heterologous 2-OG permease KgtP
from E. coli (referred as KGTP) and which can efficiently
take up 2-OG and its analogs.5,6,12,13 The uptake of
DFMPA in KGTP was studied using High Resolution
Magic Angle Spinning 19F and 1H NMR (HRMAS NMR),
an excellent nondestructive method for the in vivo
analysis of the metabolic profiles of whole cells/tissues.14
Surprisingly, DFMPA could be taken up by the KGTP
strain in a similar way to DFPA and 2-MPA, as demon-
strated by the clear signals observed for gem-difluoro-
methylene at ꢀ105 ppm in 19F NMR (Figure 1A) and
1
the corresponding vinyl group at 5.85 ppm in H NMR
respectively (Figure 1B), whereas, in the control experi-
ments, no such NMR signals were produced by the KGTP
strain or the wild type strain in the absence of DFMPA
(data not shown). The effective uptake of DFMPA by the
KGTP strain suggests its recognition by the 2-OG per-
mease and consequential transporting across the cell
membrane, thus implying that its resemblance with 2-OG
may fool the 2-OG permease. Even more surprisingly was
Synthesis of DFMPA was achieved by coupling ethyl
2-(bromomethyl)acrylate withethyl bromodifluoroacetate
in the presence of activated zinc powder and CuCN,9
followed subsequently by alkali hydrolysis (Scheme 2).
The crystal structure ofDFMPA showsa bent backbone
conformation, which differs from the extended structures
adopted by 2-OG, DFPA, and 2-MPA (Figure S1, Sup-
porting Information). This structural deviation can
be easily understood when considering the chemical
ꢀ
(10) Zhang, C.-C.; Laurent, S.; Sakr, S.; Peng, L.; Bedu, S. Mol.
Microbiol. 2006, 59, 367–375.
(6) Chen, H.; Laurent, S.; Bedu, S.; Ziarelli, F.; Chen, H. L.; Zhang,
C.-C.; Peng., L. Chem. Biol. 2006, 13, 849–856.
(7) Zhao, M. X.; Jiang, Y. L.; He, Y. X.; Chen, Y. F.; Teng, Y. B.;
Chen, Y. X.; Zhang, C.-C.; Zhou, C. Z. Proc. Natl. Acad. Sci. U.S.A.
2010, 107, 12487–12492.
(11) Meeks, J. C.; Elhai, J. Microbiol. Mol. Biol. Rev. 2002, 66, 94–
121.
ꢀ
(12) Li, J. H.; Laurent, S.; Konde, V.; Bedu, S.; Zhang, C.-C.
Microbiology 2003, 149, 3257–3263.
ꢀ
ꢀ
(13) Vazquez-Bermudez, M. F.; Herrero, A.; Flores, E. J. Bacteriol.
(8) Herrero, A.; Muro-Pastor, A. M.; Valladares, A.; Flores, E.
FEMS Microbiol. Rev. 2004, 28, 469–487.
(9) Tomoko, K.; Tomoya, K. J. Fluorine Chem. 1998, 88, 99–103.
2000, 182, 211–215.
(14) Griffin, J. L.; Pole, J. C. M.; Nicholson, J. K.; Carmichael, P. L.
Biochim. Biophysi. Acta. 2003, 1619, 151–158.
Org. Lett., Vol. 13, No. 11, 2011
2925