Total synthesis, assignment of absolute stereochemistry, and structural revision of chlorofusin

Article abstract of DOI:10.1021/ja072225g

The first total synthesis of chlorofusin was accomplished in a convergent fashion. With all four unambiguous diastereomeric model chromophores as references, the absolute stereochemistry of the chlorofusin chromophore was determined and revised to be (4S,

Full text of DOI:10.1021/ja072225g

Published on Web 05/02/2007
Total Synthesis, Assignment of Absolute Stereochemistry, and Structural
Revision of Chlorofusin
Wen-Jian Qian, Wan-Guo Wei, Yong-Xia Zhang, and Zhu-Jun Yao*
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
Received March 30, 2007; E-mail: yaoz@mail.sioc.ac.cn
Chlorofusin (1, Figure 1), a novel fungal peptide isolated from
the fermentation broth of Microdochium caespitosum in 2001,1 has
been found to antagonize p53-MDM2 interactions at micromolar
concentrations, by directly binding to the N-terminal domain of
MDM2.2 Association of phosphoprotein p53 with the oncogene
product MDM2 results in formation of a stable complex that blocks
the DNA-binding domain of p53. This renders p53 incapable of
activating transcription of the genes required for induction of G1
arrest or apoptosis.3 Therefore, by disrupting these protein-protein
interactions, chlorofusin represents an attractive lead for anticancer
therapy developments. On the basis of the spectroscopic evidence,
chlorofusin was proposed to have a densely functionalized chro-
mophore linked through the terminal amine of an ornithine residue
to a 27-membered cyclic peptide (composed of nine amino acid
residues). The absolute stereochemistry of the peptide was assigned
through the total synthesis of both possible cyclopeptides by Boger
and Searcey in 2003.4 Relying on our previous studies of the
chlorofusin chromophore,5-6 we detail herein the first total synthesis
of chlorofusin, together with a revision of its previously reported
Figure 1. Retrosynthetic analysis of chlorofusin (1).
structure and assignment of the absolute stereochemistry.
With a consideration toward convergence, chlorofusin is discon-
nected into an azaphilone (2) and cyclic peptide (3) components
(Figure 1). Coupling of these two constituents can be achieved
utilizing our previously developed conditions for the highly efficient
conjugation of azaphilone with primary amines.5 Elaboration of the
azaphilone spiro-aminal functionality is approached through in-
tramolecular bromo-etherification combined with an in situ hy-
drolysis. This provides for a fast-assembly that has proven to be
highly efficient.
Scheme 1 a
Synthesis of racemic 2 was previously reported by us.6a Prepara-
tion of (S)-2 started from the known aldehyde 4 (Scheme 1).6b
Copper-mediated asymmetric oxidation8 of 4 with 1.1 equiv of Cu2-
[(-)-sparteine]2O2 followed by treatment of aqueous NH4Cl in CH3-
CN afforded azaphilone 6 (55%, 91% ee). The tertial alcohol in 6
was then converted to its butanoate ester 7 (70%). Selective
chlorination of 7 was carried out by treatment with SO2Cl2 at room
temperature. Removal of acetate in 8 was accomplished using K2-
CO3 in MeOH, affording (S)-2 in 80% yield.
a Reaction conditions: (a) (-)-sparteine, O2, Cu(CH3CN)4PF6, DIEA,
DMAP, CH2Cl2; (b) CH3CN, aq NH4Cl, 55%, 91% ee (two steps); (c)
nPrCOCl, catalyst DMAP, pyridine, CH2Cl2, 70%; (d) SO2Cl2, CH2Cl2, room
temp, 66%; (e) K2CO3, MeOH, 80%.
Scheme 2 a
Coupling of (S)-2 with the peptide segment 3b was performed
in facile fashion employing our previously developed conditions,5
giving 9a in 80% yield (Scheme 2). The parallel reaction of rac-2
with 3b afforded two HPLC-separable diastereomers (9a and 9b,
1:1). 1H NMR of these two compounds showed a significant
difference in the chemical shifts of their C-13 methyls. Of the two
products, (4S)-9a (δ 1.43) provided a good match with the natural
product (δ 1.41), while (4R)-9b was different (δ 1.29). This suggests
that the stereogenic center at the C-4 position of chlorofusin should
be the S configuration.
a Reaction conditions: (a) aq NaHCO3, MeCN, room temp, 1 h, 80%.
ical control. Considering the insufficiency of reliable methodology
to direct the stereochemical outcome of spiro-aminal formation, it
was decided to make use of the reversible ring-opening/-closing
properties frequently observed in similar cases, and allow the natural
peptide environment of chlorofusin to affect the final formation of
its spiro aminal in a favorable direction. Toward this end, we sought
to optimize our previous methodology,6 in which bromoetherifi-
The most demanding aspect of this total synthesis is the
elaboration of the spiro-aminal moiety under favorable stereochem-
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10.1021/ja072225g CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 3 a
Figure 2. A proposed mechanism for conversion of 11d to 11a.
To avoid this side reaction, direct treatment of (4S)-2 with free
peptide 3a was employed, successfully affording the expected
compound 1a (25% after purification by HPLC). The spectroscopic
data of 1a match those reported for the natural sample. To confirm
these results, reaction of rac-2 with free peptide 3a was also
examined, giving a 1:1 mixture of 1a and 14. The 1H NMR
spectrum of this mixture showed distinct chemical shift differences
in the chromophore region as comparison to pure 1a (see Supporting
Information). On the basis of the above evidence, the configuration
of the chlorofusin chromophore1 was revised as (4S,8R,9S)-1a.
In summary, the first total synthesis of chlorofusin was ac-
complished in a convergent fashion. By comparison with all four
unambiguous diastereomeric model chromophores, the absolute
stereochemistry of the chlorofusin chromophore was finally deter-
mined as (4S,8R,9S). This allows the complete structure of natural
chlorofusin to be assigned for the first time. The enantioselective
copper-mediated oxidation of 4 followed by mild coupling of
azaphilone 2 with the amine-bearing cyclopeptide 3 in a cascade
fashion, and a final one-pot spiro-aminal formation were achieved
with high efficiency to yield the correct stereochemical product.
Further biological evaluation of chlorofusin and its isomers, as well
as synthetic model chromophores, are underway in this laboratory
and will be reported in due course.
a Reaction conditions: (a) NBS, CH3CN, room temp; (b) MeNH2HCl,
aq NaHCO3, CH3CN, room temp; (c) Ag2O, MeCN, room temp, 70% (11a:
11b:11c:11d )1:1:2.5:4, by HPLC analysis).
Scheme 4 a
a Reaction conditions: (a) (4S)-2 and 3a or 3b, same conditions as in
i
Scheme 3; (b) TFA-H2O (10: 1), Pr3SiH (2 equiv), room temp.
cation and subsequent in situ mild hydrolysis serve as the key
reactions to quickly generate a hydroxylated spiro aminal. Using
rac-2 and rac-10 as substrates, various conditions were examined
(Scheme 3). In most cases, all four possible products (11a-d)
were generated in different yields and ratios. These four sep-
arable compounds were unambiguously characterized by X-ray
single crystal analyses.6a By comparison of NMR data, 11d
was found to be the closest to the chromophore of chlorofusin
(see the Supporting Information). This indicated that the orig-
inally proposed stereochemical assignment for the chromophore
might need to be revised. To confirm our initial findings, work
focused on the synthesis of 11d. Eventually, one-pot se-
quential treatment of rac-2 in MeCN with NBS, MeNH2, and Ag2O
was found to work best, both in terms of the product ratio and-
the overall yield. This sequential treatment protocol greatly re-
duced the complexity of handling a complex molecule as
large as chlorofusin and presented the advantage of ease of
operation.
Encouraged by these results, coupling of (4S)-2 with peptide 3b
was initially attempted using a sequential protocol, giving the
expected compound 12 in 23% yield (Scheme 4). Unfortunately,
final trityl-deprotection using TFA-H2O (10:1) in the presence of
iPr3SiH (2 equiv) afforded the unexpected stereoisomer of chloro-
fusin, 13 (80%). Evidently, the spiro aminal in 12 opened and
reformed under the acidic conditions used. This was confirmed
when it was shown that rac-11d could be fully converted to rac-
11a under similar conditions (detection by HPLC). A mechanism
was proposed in Figure 2.
Acknowledgment. This project is financially supported by the
NSFC (Grants 20425205, 20432020, 20621062), CAS (Grant
KJCX2-YW-H08), and SHMCST (Grants 06QH14016, 04DZ14901).
Supporting Information Available: Experimental procedures and
characterizations of new compounds, 1H NMR and 13C NMR spectra,
and tables of NMR data and HPLC comparisons and CIF files for
compounds 11a-d. This material is available free of charge via the
Internet at http://pubs.acs.org.
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