C O M M U N I C A T I O N S
Scheme 2 a
and colleagues, that NRPS cyclization domains are somewhat
promiscuous and do not make specific contacts with the entire
peptide chain.11
Assuming that the biosynthesis of trans,trans- and cis,cis-
ceratospongamide is under kinetic control, it was of interest to
determine their relative thermodynamic stabilities. Gerwick and
colleagues reported that cis,cis-ceratospongamide could be con-
verted into the trans,trans-conformer by heating in DMSO at 175
°C. However, their HPLC data showed at least eight additional
peaks, with the trans,trans-conformer being a minor component.3
In striking contrast, we found that cis,cis-ceratospongamide 2 was
smoothly transformed (175 °C, degassed d6-DMSO) into a 5:1
equilibrium mixture favoring trans,trans-ceratospongamide 1 (see
Supporting Information). By subjecting pure 1 to the same
conditions, we again obtained a 5:1 ratio of conformers. This
experiment suggests that the thermodynamic ratio is the inverse of
the kinetic and biosynthetic ratios. In addition, it provides a high-
yielding route for the synthesis of 1.
Our synthetic route is amenable to the preparation of suitable
quantities of both ceratospongamide conformers for further biologi-
cal testing. Future studies will be directed toward elucidating the
mechanism by which trans,trans-ceratospongamide blocks IL-1â-
mediated gene expression as well as defining the structural elements
responsible for ceratospongamide’s unique conformational proper-
ties. Adapting our route to the solid phase will facilitate the
preparation of combinatorial libraries of cell-permeable cyclic
peptides with novel biological activities.
a Reagents and conditions: (a) HCl, dioxane, CH2Cl2 (b) Fmoc-OSuc,
DIPEA, THF (c) Deoxo-Fluor, CH2Cl2, -78 °C rt (100% for three steps)
(d) LiOH, MeOH, H2O (d) BOP, DMAP, syringe pump addition (depicted
yields of 1 and 2 are of HPLC-purified material).
of the crude reaction mixture. These results are in accord with those
obtained by the Shioiri group, who prepared 2 by a similar route.10
If macrocyclization to form 7, rather than oxazoline formation,
were the conformer-determining step, then closing the macrocycle
at a different amino acid position might provide the trans,trans-
conformer. In d6-DMSO, 7 gives a single set of sharp peaks,
indicating a single conformer or rapid interconversion between
multiple conformers. In contrast, there are at least three conformers
in CDCl3 (see Supporting Information). These observations dem-
onstrate that with cyclic peptide 7, conformer interconversion occurs
at room temperature. Thus, cyclization of any linear peptide to give
intermediate 7 would provide the same, thermodynamic ratio of
conformers, regardless of the reaction conditions or the position of
ring closure.
We therefore investigated an alternative strategy of closing the
macrocycle after forming the oxazoline, reasoning that the transition
state of the macrocyclization step would be significantly different
from the transition state of the oxazoline-forming reaction that
provided 2. Because the oxazoline moiety is unstable to acid, the
Boc group in 6 was switched to an Fmoc group before treatment
with Deoxo-Fluor (Scheme 2). The Fmoc-protected oxazoline 8
was thus obtained in quantitative yield. Simultaneous deprotection
of both termini with LiOH, followed by macrocyclization with BOP/
DMAP/DMF afforded a 1:10 ratio of trans,trans- and cis,cis-
ceratospongamide in 55% overall yield. Trans,trans-ceratospon-
gamide was easily purified by HPLC and was identical in all
respects to naturally derived 1 (1H and 13C NMR, HPLC co-
injection). To improve the yield of 1, we investigated alternative
cyclization conditions. Changing the solvent from DMF to a mixture
of DMF and CH2Cl2 increased the trans,trans:cis,cis ratio to 1:3
(17% HPLC-purified yield of 1), which is similar to the ratio of
conformers that were isolated from the marine sponge.3 Closing
the macrocycle at the isoleucine-proline junction in either DMF
or DMF/CH2Cl2 provided a 1:10 conformer ratio.
Acknowledgment. We thank the Sandler Family Foundation
and the Howard Hughes Medical Institute (Research Resources
Program Grant 53000284) for generous financial support.
Supporting Information Available: Experimental procedures and
spectral data for all new compounds (PDF). This material is available
References
(1) Cane, D. E.; Walsh, C. T.; Khosla, C. Science 2001, 282, 63-68.
(2) Takeuchi, Y.; Marshall, G. R. J. Am. Chem. Soc. 1998, 120, 5363-5372.
(3) Tan, L. T.; Williamson, R. T.; Gerwick, W. H.; Watts, K. S.; McGough,
K.; Jacobs, R. J. Org. Chem. 2000, 65, 419-425.
(4) For structural studies on oxazoline- and thiazole-containing cyclic peptides,
see: (a) Abbenante, G.; Fairlie, D. P.; Gahan, L. R.; Hanson, G. R.;
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It is remarkable that macrocyclization of the oxazoline-containing
peptide provided a kinetic distribution of conformers that was
virtually identical to the biosynthetic ratio, whereas formation of
the oxazoline ring after macrocyclization failed to give a trace of
the trans,trans-conformer. Ceratospongamide biosynthesis is most
likely mediated by a nonribosomal peptide synthetase (NRPS). The
fact that ceratospongamide biosynthesis produces a mixture of
conformers is consistent with the idea, first established by Walsh
(9) The unidentified minor component can be detected in the 2D, but not the
1D NMR spectra of 2 in ref 3. Thus. conformer 2 may slowly isomerize
to a third conformational isomer.
(10) Shioiri and colleagues did not report the presence of a minor conforma-
tional isomer in their synthesis of 2 (see ref 5).
(11) (a) Trauger, J. W.; Kohli, R. N.; Mootz, H. D.; Marahiel, M. A.; Walsh,
C. T. Nature 2000, 407, 215-218. (b) Kohli, R. M.; Trauger, J. W.;
Schwarzer, D.; Marahiel, M. A.; Walsh, C. T. Biochemistry 2001, 40,
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