C O M M U N I C A T I O N S
1
5
forming the resulting ureido dipeptide. Alternatively, in the
cyclization” pathway, the N-carboxy intermediate would cyclize
Velcade and presents an opportunity to expand the proteasome
inhibitor class of cancer therapeutics. We anticipate that explora-
tion of the promiscuity inherent to SylC, as evidenced by
formation of the unnatural L-allo-Ile-containing ureido-dipeptide,
should allow for the production of new syringolin analogues.
“
intramolecularly, to generate a highly reactive covalently tethered
thiohemiacetal intermediate, III. This transient thiohemiacetal
would partition in one of at least two ways. In one scenario,
1
1
formation of a “Leuch’s anhydride” of type IV would persist
as a noncovalently bound species in the SylC active site during
activation and loading of the second Val (Figure 4, pathway a).
The nucleophilic amine of the second Val-S-T intermediate
would capture IV regiospecifically and unravel it to the ureido-
Acknowledgment. This work was supported by NIH Grant
GM49338 (C.T.W.). The authors would like to thank Prof. S.
Lindow at UC-Berkeley for supplying the B728a strain of P.
syringae and Prof. R. Dudler at the University of Zurich for
discussion of results prior to publication.
1
2
peptidyl-S-SylC. Alternatively, the transient thiohemiacetal III
would be intercepted first by a nucleophilic SylC residue (Nu)
1
3
to form a covalent adduct V (Figure 4, pathway b). This in
turn would be captured by the nucleophilic amine of the second
tethered Val yielding the ureido dipeptide. Presumably, the
ureido-peptidyl-S-SylC serves as the upstream intermediate that
is transferred to the next NRPS module during chain elongation
in syringolin biosynthesis.
Supporting Information Available: Supplemental figures, ex-
perimental procedures, and HRMS characterization. This material
is available free of charge via the Internet at http://pubs.acs.org.
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(
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1
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(
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3
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of bicarbonate/CO
2
by cyclization of an initial N-carboxy-
(12) Leuch anhydrides have been utilized as peptide coupling reagents; however
no dipeptide or resulting diketopiperazines were observed as side products
from SylC incubations by HRMS.
aminoacyl-S-Ppant enzyme intermediate.
Future studies will be directed at deducing evidence in favor
of these or alternate mechanisms for formation of the ureido
group and evaluating parallel systems for the peptide chain
reversal in pacidamycins and anabaenopeptins. The mode of
action of syringolin A is similar to that of the anticancer drug
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(
(
14) O-Enrichment represented by the filled oxygens.
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4
51.
JA909170U
J. AM. CHEM. SOC. 9 VOL. 131, NO. 51, 2009 18265