Total synthesis, stereochemical reassignment, and absolute configuration of chlorofusin

Article abstract of DOI:10.1021/ja073264v

Chlorofusin is a recently isolated, naturally occurring inhibitor of p53-MDM2 complex formation whose structure is composed of a densely functionalized azaphilone-derived chromophore linked through the terminal amine of ornithine to a nine-residue cyclic peptide. Herein we report full details of the total synthesis of chlorofusin, resulting in the assignment of the absolute stereochemistry and reassignment of the relative stereochemistry of the complex chromophore. Condensation of each enantiomer of an azaphilone chromophore precursor with the N^δ-amine of a protected ornithine-threonine dipeptide, followed by a one-step oxidative spirocyclization of the most reactive olefin provided all eight diastereomers of the fully elaborated chromophore-dipeptide conjugate. Comparison of the spectroscopic properties for these eight compounds with simpler models and with that reported for the natural product allowed the full assignment of the (4R,8S,9R)-stereochemistry of the chlorofusin chromophore. The desired, but stereochemically reassigned, diastereomer of the dipeptide conjugate was incorporated in a convergent total synthesis of chlorofusin confirming both the relative stereochemical reassignment and the establishment of its absolute stereochemistry. In addition to natural chlorofusin, the (4R,8R,9R)-diastereomer proposed by Williams as well as the (4R,8S,9S) and (4R,8R,9S)-diastereomers of chlorofusin were also prepared as key analogues of the natural product, and the noncorrelation of their spectral data with that reported for natural chlorofusin further supports the new structural assignment and maintains the diagnostic spectroscopic distinctions observed with the eight diastereomers of the chromophore ornithine-threonine dipeptide conjugates. The reassignment of the relative stereochemistry for the chlorofusin chromophore is still, but less obviously, consistent with the experimental NOEs observed by Williams in the original structural assignment work, and the assignment of its absolute configuration is opposite that recently disclosed by Yao. Copyright

Full text of DOI:10.1021/ja073264v

Published on Web 07/19/2007
Total Synthesis, Stereochemical Reassignment, and Absolute Configuration
of Chlorofusin
Sang Yeul Lee, Ryan C. Clark, and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received May 8, 2007; E-mail: boger@scripps.edu
Chlorofusin (1, Figure 1A) was isolated from the fungal strain
Microdochium caespitosum and found to disrupt the MDM2-p53
interaction by binding to the N-terminal domain of MDM2.1 As
such, chlorofusin represents an exciting lead for antineoplastic
intervention that acts by a rare disruption of a protein-protein
interaction.2 On the basis of spectroscopic and degradation studies,
the structure was proposed to be composed of a densely function-
alized chromophore linked through the terminal amine of ornithine
to a nine-residue cyclic peptide.1 Although the studies permitted
the identification of the cyclic peptide structure and connectivity,
the two asparagine residues were only established to have opposite
stereochemistries (L and D), and their respective assignments were
not possible. Previously, we reported the synthesis of the two cyclic
peptide diastereomers bearing either the L-Asn3/D-Asn4 or D-Asn3/
L-Asn4 stereochemistry and were able to correlate the former with
the spectroscopic properties of the natural product.3
Similarly, the spectroscopic studies conducted by Williams
provided an assigned relative stereochemistry for the chromophore,
but did not permit an assignment of its absolute stereochemistry
(Figure 1B). The relative stereochemistry was assigned using
gradient 1D NOE studies albeit entailing a long-range C4-Me/C8-H
Figure 1. (A) Chlorofusin; (B) previous chromophore assignments.
Scheme 1
NOE observed only at very extended mixing times (500 ms).
Prompted by the recent disclosure of Yao that claims to have
prepared chlorofusin,4 herein we report our independent reassign-
ment of the chromophore relative stereochemistry that is still, but
less obviously, consistent with the experimental NOEs reported by
Williams and an assignment of its absolute configuration that is
opposite that disclosed by Yao. A total synthesis of this revised
chlorofusin structure provided material displaying spectroscopic
properties indistinguishable from that reported for the natural
product confirming the new chromophore structural assignment and
establishing the accuracy of our earlier L-Asn3/D-Asn4 cyclic
peptide assignment.
Since the absolute configuration of the chromophore was
unknown, the route pursued in our first generation synthesis
permitted access to both enantiomeric series and to all possible
diastereomers, albeit developed to access the Williams assigned
diastereomer depicted in Figure 1B. The azaphilone 2 was prepared
following established protocols5 and was chromatographically
resolved (Daicel CHIRALCEL OD column, 2 × 25 cm, 20%
EtOH-hexanes, 7 mL/min; tR 22.2 min (S)-2, 25.0 min (R)-2) into
its enantiomers. Their absolute configurations were assigned on the
basis of the diagnostic sign of the longest wavelength (350-370
nm) Cotton effect in their CD spectra6a which, for such simple
azaphilones, empirically has been shown to also correlate with the
similarly diagnostic sign of their optical rotation.6b The Nδ-amine
of ornithine in dipeptide 3, constituting the L-Orn-L-Thr segment
of the cyclic peptide, was condensed with azaphilones (R)-2 or (S)-2
to provide 4 and 5 (Scheme 1). Subjection of each to a single-step
oxidative spiroketalization (I2, AgNO3, H2O-DMSO) of the C8-
C9 double bond provided all four diastereomers of the two
enantiomeric series. Although space precludes a discussion of the
development of this protocol, it is initiated by reversible iodonium
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10.1021/ja073264v CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Synthesis of Chlorofusin
the carboxylic acid of heptapeptide 15 cleanly provided 16 (EDCI,
HOAt, NaHCO3, DMF, 0-23 °C, 24 h, 55%). Simultaneous benzyl
ester deprotection and Cbz removal (H2, Pd/C, THF-DMF, 4 h)
provided the corresponding amino acid 17 which was cyclized upon
treatment with EDCI-HOAt (NaHCO3, DMF, 0-23 °C, 40 h,
60%) to provide material with spectroscopic properties indistin-
guishable from that reported for chlorofusin. In addition to natural
chlorofusin, the (4R,8R,9R)-diastereomer proposed by Williams and
the (4R,8S,9S) and (4R,8R,9S)-diastereomers of chlorofusin were
also prepared by this route from 6-8, and the anticipated noncor-
relation of their spectroscopic properties with that reported for the
natural product provided further support for the new structural
assignment.
This (4R,8S,9R)-diastereomer (1) is clearly distinguishable from
the diastereomer reported by Yao who correctly reassigns the
chromophore relative stereochemistry, but which possesses the
wrong absolute stereochemistry. His (4S,8R,9S)-diastereomer (the
chromophore enantiomer), even with his adjusted chemical shifts,
exhibited the diagnostic ornithine CH2δ signal as two multiplets of
1H each (δ 3.42 and 3.50) analogous to 13 (vs δ 3.42, t, 2H for
natural1 and synthetic chlorofusin) making it readily distinguishable
from the data reported for the natural product.8
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (Grant CA41101 and
CA42056) and the Skaggs Institute for Chemical Biology. We
especially thank Dr. S. S. Pfeiffer for initial studies on the
chromophore synthesis and Dr. S. S. Pfeiffer and Dr. P. Desai for
the original studies on the chlorofusin cyclic peptide.3 S.Y.L. and
R.C.C. are Skaggs Fellows.
ion formation and subsequent iodoetherification with N,O-ketal
formation followed by Ag(I)-assisted displacement of the iodide
by H2O-DMSO providing 6-13 directly. The major products, in
which the C8 and C9 oxygen substituents are syn possessing the
C8/C9 stereochemistry found in the Williams assignment, represent
those that formally arise from a trans iodoetherification reaction
followed by SN2 displacement of the iodide by water. Analogous
to and extending unambiguous stereochemical assignments (X-ray
and interconversion studies) made first with model benzylamine
and n-butylamine azaphilone adducts (Supporting Information), the
structures of all eight diastereomers were fully assigned using
COSY, HMQC, HMBC, and ROESY NMR. The two syn and two
anti diastereomers in each enantiomeric series are readily distin-
guishable by diagnostic 1H NMR (C10-H, C12-H and C8-OH) and
13C NMR (C1, C2,7 C6,7 C10, and C12) chemical shifts, and the
two anti diastereomers within each enantiomeric series are most
Supporting Information Available: Full experimental details are
provided. This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) (a) Duncan, S. J.; Grueschow, S.; Williams, D. H.; McNicholas, C.;
Purewal, R.; Hajek, M.; Gerlitz, M.; Martin, S.; Wrigley, S. K.; Moore,
M. J. Am. Chem. Soc. 2001, 123, 554-560. (b) Duncan, S. J.; Cooper,
M. A.; Williams, D. H. Chem. Commun. 2003, 316-317.
(2) Boger, D. L.; Desharnais, J.; Capps, K. Angew. Chem., Intl. Ed. 2003,
42, 4138-4176.
(3) (a) Desai, P.; Pfeiffer, S. S.; Boger, D. L. Org. Lett. 2003, 5, 5047-
5050. For additional preparations of this cyclic peptide, see: (b) Malkinson,
J. P.; Zloh, M.; Kadom, M.; Errington, R.; Smith, P. J.; Searcey, M. Org.
Lett. 2003, 5, 5051-5054. (c) Tomonori, M.; Miyagi, M.; Suzuki, K.;
Shibasaki, M.; Saikawa, Y.; Nakata, M. Heterocycles 2007, 72, 275-
291.
(4) Qian, W.-J.; Wei, W.-G.; Zhang, Y.-X.; Yao, Z.-J. J. Am. Chem. Soc.
2007, 129, 6400-6401.
1
readily distinguished by diagnostic C8-H, C13-H, and C8-OH H
NMR and C7 and C13 13C NMR chemical shifts. Supporting the
assignments were N,O-ketal equilibration studies which relate syn/
anti diastereomer pairs.
(5) Chong, R.; King, R. R.; Whalley, W. B. Chem. Commun. 1969, 1512-
1513. For additional studies on azaphilones related to the chlorofusin
chromophore, see: Zhu, J.; Grigoriadis, N. P.; Lee, J. P.; Porco, J. A., Jr.
J. Am. Chem. Soc. 2005, 127, 9342-9343. Wei, W.-G.; Qian, W.-J.;
Zhang, Y.-X.; Yao, Z.-J. Tetrahedron Lett. 2006, 47, 4171-4174.
(6) (a) Steyn, P. S.; Vleggaar, R. J. Chem. Soc., Perkin Trans. 1 1976, 204-
206. (b) Whalley, W. B.; Ferguson, G.; Marsh, W. C.; Restivo, R. J. J.
Chem. Soc., Perkin Trans. 1 1976, 1366-1369.
Out of this set of eight diastereomers, the (4R,8S,9R)-diastere-
omer 9 provided a near perfect match with the spectroscopic
properties reported for the chlorofusin chromophore, whereas the
(4S,8R,9S)-diastereomer 13 (chromophore enantiomer) proved
(7) These two assignments (δ 115.2 for C2, 101.3 for C6 as reported by
Williams) may be switched (δ 101.3 for C2, 115.2 for C6). This tentative
reassignment is under continued investigation.
1
readily distinguishable by both the H NMR chemical shift and
(8) Subsequent to the web disclosure of ref 4 as well as following the
completion of our work, we re-examined a sample of authentic chlorofusin
provided by Dr. Stephen Wrigley (2003, but aged and of unknown quality)
that failed to provide a discernable 1H NMR spectrum at that time. With
an intimate knowledge of the chromatographic and physical properties of
such compounds in hand, the processing of the remaining material (<1
mg) provided a sample that exhibited a CD spectrum indistinguishable
(sign and magnitude) from synthetic 1 confirming our absolute configu-
ration assignment and an 1H NMR spectrum of sufficient quality to confirm
that it represents the authentic natural product (see Supporting Information).
multiplicity of the ornithine CH2δ adjacent to the chromophore (δ
3.45, m, 2H for 9 vs δ 3.41 and 3.52, two m, 1H each for 13;
chlorofusin ) δ 3.42, t, 2H). This final multiplicity distinction
allowed the absolute stereochemical assignment for the chro-
mophore.
Accordingly, the (4R,8S,9R)-diastereomer 9 was incorporated into
a total synthesis of chlorofusin. Fmoc deprotection (piperidine,
CH2Cl2-DMF, 40 min) and coupling of the free amine 14 with
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