In vitro Inhibition Studies of Phytoene Desaturase
J. Agric. Food Chem., Vol. 49, No. 1, 2001 141
LITERATURE CITED
contrast, the most potent phytoene desaturase inhibi-
tors, diflufenican, fluridone, and flurtamone, exhibit I50
values between 10-8 and 10-9 M.
(1) Bo¨ger, P.; Sandmann, G. Carotenoid biosynthesis inhibi-
tor herbicides - mode of action and resistance mecha-
nisms. Pestic. Outlook 1998, 9, 29-35.
(2) Chamovitz, D.; Sandmann, G.; Hirschberg, J . Molecular
and biochemical characterization of herbicide-resistant
mutants of cyanobacteria reveals that phytoene desatu-
ration is a rate-limiting step in carotenoid biosynthesis.
J . Biol. Chem. 1993, 268, 17348-17353.
(3) Sandmann, G.; Fraser, P. D. Differential inhibition of
phytoene desaturase from diverse origins and analysis
of resistant cyanobacterial mutants. Z. Naturforsch.,
C: J . Biosci. 1993, 48c, 307-311.
(4) Martinez-Ferez, I.; Vioque, A.; Sandmann, G. Mutagen-
esis of an amino acid responsible in phytoene desaturase
from Synechocystis for binding of the bleaching herbicide
norflurazon. Pestic. Biochem. Physiol. 1994, 48, 185-
190.
For the homochiral derivatives A, B, and C, inhibition
of phytoene desaturase appears to be rather more
dependent on the stereochemistry of the ketomorpholine
5-methyl substituent than that of the 2-(3-trifluorom-
ethylbenzyl) substituent; compound A [(2R), (5S)-
isomer] is the most active, followed by compound C [(2S),
(5S)-isomer], with compound B [(2S), (5R)-isomer] being
the least active. The enhanced activity of the trans-
isomer (compound A) over the cis-isomer (compound C)
was predicted by our earlier model for the herbicide
binding site, which was constructed on the basis of
whole plant herbicidal activity (7) and was now estab-
lished for interaction of the ketomorpholines with the
target enzyme.
The in vitro inhibition of the racemic N-methyl
derivative (compound H) is significantly reduced relative
to the corresponding N-H derivative (compound D). By
comparison of the in vitro activities of compounds D, E,
and G, it is clear that the distance of the substituted
phenyl group from the ketomorpholine ring is an
important factor for their interaction with phytoene
desaturase. The influence of either -CH2- or -S- as
a linking group is very similar, but -CH2S- as a linking
group significantly increased phytoene desaturase in-
hibition of compound G. This derivative is isosteric with
the CH2-CH2-linked compound which was predicted in
our earlier modeling work (based on overall herbicidal
activity) to be a less-active herbicide than compound D
(7). The difference may be explained by a better interac-
tion of compound G via the sulfur group in addition to
the negative steric effect of the extended bridge.
Recently, a good correlation between inhibition of
phytoene desaturase and preemergence herbicidal ac-
tivity for a series of bleaching 3-trifluoromethyl-1,1′-
biphenyl derivatives assayed with two plant species was
reported (13). This is also the case for our ketomorpho-
line derivatives (Figure 3) when considering the average
herbicidal score obtained with 7 different weed species.
However, compounds G and H did not fit into the
correlation. In whole plants, compound G is less herbi-
cidal than expected from its phytoene desaturase I50
value. One explanation may be in vivo oxidation of this
compound to a less potent sulfoxide or sulfone deriva-
tive: we know that the corresponding sulfides (mixture
of diastereoisomers) and sulfone of compound E are
inactive as herbicides (Mitchell et al., unpublished). In
contrast, compound H shows rather better activity in
whole plants than might be expected from its poor in
vitro phytoene desaturase activity. Here again a modi-
fication in plants may occur, most likely N-demethyla-
tion to compound D which is a much more active
herbicide; similar activation by N-demethylation has
been reported for the bleaching herbicide metflurazon
(14). As judged from the highly variable pattern of weed
susceptibility towards compound H as compared to that
of the other ketomorpholines (e.g., Xanthium strumatum
showed a 15 to 20-fold higher ED50 value than Cassia
obtusifolia, Chenopodium album, or Galium aparine;
Table 1), the potential to modify compound H may be
different in various plants.
(5) Sandmann, G.; Bo¨ger, P. Chemical structure and activity
of herbicidal inhibitors of phytoene desaturase. In
Rational Approaches to Structure, Activity, and Ecotoxi-
cology of Agrochemicals; Draber, W., Fujita, T., Eds.;
CRC Press: Boca Ration, FL, 1992; pp 357-371.
(6) Mitchell, G.; Bartlett, D. L. 2-(3-Trifluoromethylbenzyl)-
3-ketomorpholine - a new phytoene desaturase inhibi-
tor: optimisation of in vitro activity using computer
modelling. In 7th Int. Congr. Pestic. Chem., Book of
Abstracts Vol I.; Frehse, H., Kesseler-Schmitz, E., Con-
way, S., Eds.; Poster 01D-04, Hamburg, Germany, 1990;
p 170.
(7) Mitchell, G. Phytoene desaturase. A model for the
optimization of inhibitors. In Synthesis and Chemistry
of Agrochemicals IV, Baker, D. A., Fenyes, J . G., Moberg,
W. K., Cross, B., Eds.; ACS Symposium Series, Vol. 584,
American Chemical Society: Washington, DC, 1995; pp
161-170.
(8) Pecker, I.; Chamovitz, D.; Linden, H.; Sandmann, G.;
Hirschberg, J . A single polypeptide catalyzing the
conversion of phytoene to ú-carotene is transcriptionally
regulated during tomato fruit ripening. Proc. Natl. Acad.
Sci., U.S.A. 1992, 89, 4962-4966.
(9) Sandmann, G.; Schneider, C.; Bo¨ger, P. A new nonra-
diactive assay of phytoene desaturase to evaluate bleach-
ing herbicides. Z. Naturforsch., C: J . Biosci. 1996, 51c,
534-538.
(10) Maniatis, T.; Fritsch, E. F.; Sambrook, J . Molecular
Cloning. A Laboratory Manual, 2nd ed.; Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, NY,
1982.
(11) Sandmann, G. In vitro assay system for phytoene
desaturase inhibitors with isolated thylakoids, In Target
Assays for Modern Herbicides and Related Phytotoxic
Compounds; Bo¨ger, P., Sandmann, G., Eds.; Lewis
Publishers, Boca Raton, FL, 1993; pp 15-20.
(12) Misawa, N.; Masamoto, K.; Hori, T.; Ohtani, T.; Bo¨ger,
P.; Sandmann, G. Expression of an Erwinia phytoene
desaturase gene not only confers multiple resistance to
herbicides interfering with carotenoid biosynthesis but
also alters xanthophyll metabolism in transgenic plants.
Plant J . 1994, 6, 481-489.
(13) Laber, G.; Usunow, E.; Wiecko, W.; Franke, H.; Ko¨hn,
A. Inhibition of Narcissus pseudonarcissus phytoene
desaturase by herbicidal 3-trifluoromethyl-1,1′-biphenyl
derivatives. Pest. Biochem. Physiol. 1999, 63, 173-184.
(14) Motooka, P. S.; Corbin, F. T.; Worsham, A. D. Metabo-
lism of Sandoz 6706 in soybean and sicklepod. Weed Sci.
1977, 25, 9.
ACKNOWLEDGMENT
Received for review August 24, 2000. Revised manuscript
received October 25, 2000. Accepted October 25, 2000.
The authors thank Dr. D. J . Collins and Mr. C. V.
Coles for the herbicidal activity determinations.
J F0010432