Biomedical Engineering Reference
In-Depth Information
substrates of p-glycoprotein (pGP) or multidrug resistance protein (mrp)—two efflux
pumps responsible for much of the drug resistance observed in cancer cells [57].
The biosynthesis of these compounds was recently elucidated and, as with many
of the compounds discussed thus far, they are constructed by a hybrid PKS-NRPS and
is assembled in four portions:
d
-hydroxyoctenoic acid, 3-chloro-
O
-methyl-D-tyrosine,
methyl-
b
-alanine, and L-leucic acid. The linear assembly of these four portions is then
cyclized by a thioesterase and acted upon by several tailoring enzymes [58].
Unlike many of the systems discussed here, a process for the large-scale
fermentation has yet to be developed and production levels by the native hosts are
low. As a result of these factors, a good deal of work has been invested in the total
synthesis of natural cryptophycin as well as unnatural analogues.
The synthesis of
seco
-cryptophycin analogues is well established, but the
subsequent cyclization has presented some synthetic difficulty. This led researchers
to examine the promiscuity of the cryptophycin thioesterases.
In vitro
reactions of
seco
-cryptophycin analogues with purified cryptophycin TE led to the successful
production of compounds
[58].
SAR studies have established the essential nature of the
b
-epoxide unit installed
on the
d
-hydroxyoctenoic acid unit by a P450 epoxidase CrpE. This moiety leads to an
increase in potency of several orders of magnitude and yet has proven difficult to
install by synthetic means. The reactivity of the epoxide necessitates late-stage
installation, but on these complex molecules, the best enantioselectivity achieved
is 3:1 for the
a
over the
b
form. This led researchers to attempt the epoxidation of
unnatural cryptophycins by crpE. These reactions were again performed
in vitro
and successfully led to the selective installation of the
b
-epoxide group on compounds
70
-
73
[59,60].
This use of overexpressed and purified tailoring enzymes to perform single
selected transformations on an otherwise synthetic scaffold has provided a vastly
simplified route to analogues of one of the most potent tubulin destabilizers isolated to
date. It also provides a representative example of the power of using single purified
biosynthetic enzymes to perform synthetically difficult reactions.
This approach, along with that presented for the glycosylation of unnatural
analogues of apoptolidin, is in sharp contrast to those presented earlier, where the
biosynthetic enzymes installed the bulk of the complexity. Nonetheless, they are
complementary to each other.
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-
69
14.5. CONCLUSION
Presented above are a series of examples where precursor-directed biosynthesis was
successfully applied for the production of unnatural analogues of clinically relevant
naturally products. Through these studies, many compounds have been produced that
would have presented synthetically intractable problems, and much has been learned
about the promiscuity (or lack thereof) of several biosynthetic systems.
This chapter is by no means an extensive review of the area of precursor-
directed biosynthesis. It does, however, present a series of examples that span the
continuum of biosynthetic complexity with the goal of making molecule makers
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