C O MMU N I C A T I O N S
and methylmalonyl-S-EpoC to yield the amino-alkyl-thiazolyl-
methyacrylic acid product 9 was validated by mass spectroscopy
(198.99 observed, 199.05, expected) and by co-injection with an
authentic standard (Figure 2D). Additionally, product 10, which
represents another double substrate substitution, could be produced
from Gly-S-ACP and Ser-S-EpoB as evidenced by mass spectros-
copy (183.05 observed, 183.07, expected) (Figure 2E). When the
cationic amino group of glycyl-CoA was blocked with N-Boc,
acylglycyl transfer also occurred to yield the N-blocked-amino-
alkyl-thiazolyl-methyacrylic acid product 11 (data not shown).
Transfer of N-Boc protected intermediates among the protein
interfaces appeared to be slightly slower than the corresponding
processing of the free amine (data not shown), suggesting that steric
bulk may slow substrate processing.
The first three enzymes of the epothilone biosynthetic machinery
can utilize serine to install an oxazole in place of a thiazole in the
epothilone structure. Furthermore, EpoB will tolerate functionalized
donor groups from the EpoA-ACP domain to produce epothilone
fragments modified at the C21 position. These studies with the early
enzymes of the epothilone biosynthesis cluster suggest that com-
binatorial biosynthesis may be a viable means for producing a
variety of epothilone analogues that incorporate diversity into the
heterocycle starter unit.
Acknowledgment. Funding for this work was provided by the
NIH (GM20011). T.L.S. is supported by Postdoctoral Fellowship
Grant no. PF-02-023-01-CDD from the American Cancer Society.
S.E.O. acknowledges an Irving Sigal postdoctoral fellowship
(American Chemical Society). We acknowledge Kosan Biosciences
for providing a cosmid containing the epoA, epoB, and epoC
sequences.
Figure 2. Radio-HPLC traces of EpoA/B/C reactions characterized by
comparison to UV-HPLC of chemically synthesized standards or MS data.
(
A) Formation of 4 validated by co-injection. (B) Failure of reaction of
Dap-S-EpoB to form methyl imidazole compound 7, validated by co-
injection. (C) Formation of 8, validated by MS. (D) Formation of 9, validated
by co-injection and MS. (E) Formation of 10, validated by MS.
Supporting Information Available: Details of protein assays and
chemical standard synthesis (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
(
16.5 kDa) as evidenced by monitoring the shift of radioactivity
on a protein gel using autoradiography (see Supporting Information).
Further analysis of the alkaline-hydrolyzed products with radio-
HPLC indicated that a very small amount of acetyl-Thr-S-EpoB
appeared to cyclize to give the corresponding 2,5-dimethyloxazole
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3
and [ H]-serine as well as methylmalonyl-CoA, NADPH, and EpoC.
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(
Figure 2C), represents a double substitution that can be carried
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(8) For details of MALDI mass spectroscopy and for synthesis and charac-
terization of authentic standards, refer to Supporting Information.
(
Introduction of polar functional groups at the epothilone C21
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9
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