Insect Control: Biological and Synthetic Agents - Index of

212 6: The Spinosyns: Chemistry, Biochemistry, Mode of Action, and Resistance
screening (HTS) program was established that used
a number of target insect pests including lepidopterous
larvae, such as beet armyworm (Spodoptera
exigua), in a 96-well format, as the primary natural
products screening tool. This was accompanied by a
number of secondary assays to further characterize
extracts that were found to be active in the primary
screen. One particular multiyear effort, to screen a
very large number and variety of actinomycete broth
extracts, relied on a novel bioassay as the secondary
assay. From this screening effort an extract
was found that was not only active in the primary
diet-based screen, but that, on further testing, also
exhibited interesting contact activity coupled with
symptomology that was very reminiscent of the
spinosyns. Further separation and isolation coupled
to detailed physical characterization (nuclear magnetic
resonance (NMR), liquid chromatography–
mass spectroscopy (LC–MS), etc.) of this extract
showed that the activity was indeed due to a spinosyn-like
entity (Lewer et al., 2001, 2003). However,
there were some structural differences relative to the
then known spinosyns. The dominant, distinctive
feature was the presence of a 2-butenyl group at
the C21 position of the macrocyclic ring system in
place of the heretofore typical ethyl-group of the
spinosyns (Lewer et al., 2003) (Figure 2). The primary
factor isolated from the mixture of 21-butenyl
factors was the 21-butenyl homolog of spinosyn
A(Figures 1 and 2, Table 1). To date more than 30
additional factors have been isolated, identified, and
characterized (Lewer et al., 2003). These 21-butenyl
spinosyns constitute a new family of spinosyns, for
which research on the isolation, characterization,
and chemistry is continuing (Crouse et al., 2002).
Characterization of the microorganism that produces
the 21-butenyl spinosyns showed it to be a
new species of Saccharopolyspora closely related to,
but distinct from, S. spinosa, which has been named
Saccharopolysopra pogona (Lewer et al., 2003).
6.2.3. Spinosyn Biosynthesis
The spinosyns are 12-member macrocyclic lactones
(macrolides). Based on incorporation studies using
13 C-labeled acetate, propionate, butyrate, and isobutyrate,
the biosynthesis of the tetracycle of the
spinosyns is consistent with a polyketide synthasebased
(PKS) pathway (Kirst et al., 1992). As a
macrolide, the spinosyns differ from the norm in
that the spinosyn PKS pathway ultimately leads to
the introduction of three intramolecular carbon–
carbon bonds to form the spinosyn tetracycle
(Waldron et al., 2001) (Figure 3). To this tetracycle
a neutral sugar, rhamnose, is coupled at the C9
position, and then subsequently methylated via
three O-methyltransferases. The final steps involve
the addition of an amino sugar to the tetracycle at
the C17 position. The amino sugar, forosamine, is
also methylated prior to attachment to the spinosyn
tetracycle – in this case at both positions of the
nitrogen (Kirst et al., 1992; Waldron et al., 2000,
2001) (Figure 3).
Figure 2 Structures for 21-butenyl spinosyn A (top), the spinosyn A C17-pseudoaglycone (PSA, bottom; X ¼ H) and the C17
L-mycarose biotransformation product.