Total synthesis of chlorofusin, its seven chromophore diastereomers, and key partial structures

Article abstract of DOI:10.1021/ja8012819

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 the 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 oxidation/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 and those of simpler models with that reported for the natural product allowed the full assignment of the (4R,8S,9R)-stereochemistry of the chlorofusin chromophore. The natural, but stereochemically reassigned, diastereomer of the dipeptide conjugate was incorporated in a convergent total synthesis of chlorofusin confirming the stereochemical reassignment and establishing its absolute stereochemistry. Similarly and enlisting the late stage convergent point in the total synthesis, the remaining seven diastereomers of the chromophore-dipeptide conjugates were individually incorporated into the nine-residue cyclic peptide of chlorofusin (4 steps each) providing all seven remaining possible chromophore diastereomers of the natural product.

Full text of DOI:10.1021/ja8012819

Published on Web 08/20/2008
Total Synthesis of Chlorofusin, Its Seven Chromophore
Diastereomers, and Key Partial Structures
Ryan C. Clark, Sang Yeul Lee, 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 February 20, 2008; E-mail: boger@scripps.edu
Abstract: 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 the 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 Nδ-amine of a protected ornithine-threonine dipeptide, followed by a one-
step oxidation/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 and those of simpler models with that reported for the natural product allowed the full assignment
of the (4R,8S,9R)-stereochemistry of the chlorofusin chromophore. The natural, but stereochemically
reassigned, diastereomer of the dipeptide conjugate was incorporated in a convergent total synthesis of
chlorofusin confirming the stereochemical reassignment and establishing its absolute stereochemistry.
Similarly and enlisting the late stage convergent point in the total synthesis, the remaining seven
diastereomers of the chromophore-dipeptide conjugates were individually incorporated into the nine-residue
cyclic peptide of chlorofusin (4 steps each) providing all seven remaining possible chromophore
diastereomers of the natural product.
as an attractive target for therapeutic intervention.4,17 An X-ray
crystal structure of the N-terminal domain of MDM2 bound to
Introduction
The tumor suppressor p53 is an important part of an innate
cancer defense mechanism and acts as a transcription factor that
initiates cell cycle arrest and apoptosis in response to stress such
as DNA damage.1–4 The activity of p53 is modulated by MDM2
(HDM2), which tightly binds p53 preventing it from acting as
a regulator of cell division5–7 and targeting it for nuclear export
and degradation.8,9 Overexpression of MDM2 has been impli-
cated in many cancers,10–16 defining the p53-MDM2 interaction
a 15-residue transactivation domain of p53 revealed the
structural details of their complex that is mediated by the
interaction of three hydrophobic residues of a p53 R-helix with
a hydrophobic cleft of MDM2.18 Molecules that bind the
hydrophobic cleft of MDM2 disrupt this protein-protein
interaction with p53, restoring its regulatory function and
inhibiting tumor growth.4,19–22
Chlorofusin (1, Figure 1) was isolated from the fungal strain
Microdochium caespitosum23 and found to disrupt the p53-
MDM2 interaction by directly binding to the N-terminal domain
of MDM2 (IC50 ) 4.6 µM, KD ) 4.7 µM).23,24 Thus, chlorofusin
represents an exciting lead for antineoplastic intervention that
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A R T I C L E S
Clark et al.
Figure 2. Original chromophore relative stereochemical assignment.23 No
Figure 1. Chlorofusin.
assignment of absolute stereochemistry was made.
acts by a rare disruption of a protein-protein interaction
although the structural details of this inhibitory interaction
derived from MDM2 binding have yet to be established.20,21,25
On the basis of extensive spectroscopic and degradation studies,
the chlorofusin structure was proposed to consist of a densely
functionalized, azaphilone-derived chromophore linked through
the terminal amine of ornithine to a 27-membered cyclic peptide
composed of nine amino acid residues.23 Two of the cyclic
peptide amino acids possess a nonstandard or modified side
chain, and four possess the D-configuration. Although the
spectroscopic and degradation studies of chlorofusin permitted
the identification of the cyclic peptide structure and connectivity,
the two asparagine residues Asn3 and Asn4 were only estab-
lished to have opposite stereochemistries (L and D) and their
respective assignments were not possible. In earlier studies, we
reported the synthesis of the two cyclic peptide diastereomers
bearing either the L-Asn3/D-Asn4 or D-Asn3/L-Asn4 stereo-
chemistry and correlation of the former with the spectroscopic
properties (1H and 13C NMR) of the natural product.26 In that
work, four key subunits were assembled, sequentially coupled,
and cyclized to provide the nine residue cyclic peptide. The
coupling and macrocyclization sites were chosen to minimize
the use of protecting groups and maximize the convergency of
the synthesis, and a deliberate late-stage incorporation of the
subunit bearing the two asparagine residues allowed convenient
access to both diastereomers required to assign the absolute
stereochemistry. Concurrent with this disclosure, Searcey
reported the synthesis of the L-Asn3/D-Asn4 diastereomers
incorporating either a D-ADA8 or L-ADA8 residue,27 and
recently Nakata28 has also reported a synthesis of this cyclic
peptide.
Figure 3. Stereochemical assignment made by Yao and co-workers.30
only the protons attached to C10. In addition, irradiation of the
C4-Me protons provided a long-range NOE to the proton
attached to C8, but only if conducted with very extended mixing
times (500 ms). These results suggested that the protons attached
to C8, C10 and C4-Me all lie on the same face of the
chromophore and led to the relative stereochemical assignment
depicted in Figure 2.
Herein, we report the full details of studies leading to a
reassignment of this relative stereochemistry for the chlorofusin
chromophore that is still, but less obviously, consistent with
these experimentally observed NOEs, as well as the assignment
of the chromophore absolute configuration (4R,8S,9R). A total
synthesis of this revised chlorofusin structure provided material
displaying spectroscopic properties indistinguishable from that
reported for the natural product confirming the new chro-
mophore structural assignment and establishing the accuracy
of our earlier L-Asn3/D-Asn4 cyclic peptide stereochemical
assignment. A preliminary account of the first stage of these
studies was disclosed29 immediately following the report of
Yao30 which claimed to have completed a total synthesis of
the natural product. Although this group similarly reassigned
the chromophore relative stereochemistry, they misassigned the
absolute stereochemistry overlooking key diagnostic spectro-
scopic differences that distinguish their unnatural (4S,8R,9S)-
diastereomer 4 (Figure 3) from the natural (4R,8S,9R)-
diastereomer. In extending our previously disclosed studies
which provided all four 4R-diastereomers of chlorofusin, we
have now additionally incorporated the four 4S-diastereomers
of the chlorofusin chromophore (enantiomeric series) into fully
elaborated chlorofusin diastereomers. Thus, in addition to natural
chlorofusin, the remaining seven chlorofusin chromophore
diastereomers [(4R,8R,9R), (4R,8S,9S), (4R,8R,9S), (4S,8S,9S),
(4S,8R,9R), (4S,8S,9R), and (4S,8R,9S)] including the (4R,8R,9R)/
(4S,8S,9S)-diastereomers proposed by Williams as well as the
Similarly, the chlorofusin spectroscopic studies conducted by
Williams provided the structure and an assigned relative
stereochemistry for the unusual azaphilone-derived chro-
mophore, but did not permit an assignment of its absolute
stereochemistry (Figure 2). The relative stereochemistry was
assigned using gradient 1D NOE studies in which irradiation
of the proton attached to C8 with a mixing time of 50 ms
resulted in NOEs to the protons attached to C10 as well as C12,
whereas a shorter mixing time of 25 ms resulted in NOEs to
(25) Boger, D. L.; Desharnais, J.; Capps, K. Angew. Chem., Int. Ed. 2003,
42, 4138.
(26) Desai, P.; Pfeiffer, S. S.; Boger, D. L. Org. Lett. 2003, 5, 5047.
(27) (a) Malkinson, J. P.; Zloh, M.; Kadom, M.; Errington, R.; Smith, P. J.;
Searcey, M. Org. Lett. 2003, 5, 5051. For additional studies, see (b)
Woon, E. C. Y.; Arcieri, M.; Wilderspin, A. F.; Malkinson, J. P.;
Searcey, M. J. Org. Chem. 2007, 72, 5146.
(29) Lee, S. Y.; Clark, R. C.; Boger, D. L. J. Am. Chem. Soc. 2007, 129,
9860.
(30) (a) Qian, W.-J.; Wei, W.-G.; Zhang, Y.-X.; Yao, Z.-J. J. Am. Chem.
Soc. 2007, 129, 6400. For preceding studies see (b) Wei, W.-G.; Zhang,
Y.-X.; Yao, Z.-J. Tetrahedron 2005, 61, 11882. (c) Wei, W.-G.; Yao,
Z.-J. J. Org. Chem. 2005, 70, 4585. (d) Wei, W.-G.; Qian, W.-J.;
Zhang, Y.-X.; Yao, Z.-J. Tetrahedron Lett. 2006, 47, 4171.
(28) Mori, T.; Miyagi, M.; Suzuki, K.; Shibasaki, M.; Saikawa, Y.; Nakata,
M. Heterocycles 2007, 72, 275.
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Total Synthesis of Chlorofusin
A R T I C L E S
Scheme 1
Yao misassigned (4S,8R,9S)-diastereomer were synthesized as
key analogues of the natural product and the comparison of their
spectral properties with that reported for chlorofusin provide
unambiguous support for the structural reassignment.
scopic distinctions (1H and 13C NMR) made possible by access
to all diastereomers. Due to concerns of an unambiguous
stereochemical assignment as the studies progressed, the strategy
of early stage versus late stage chromophore oxidative spiro-
cyclization with clear stereochemical assignments for all dia-
stereomers became the focus of our efforts and was effectively
conducted on the azaphilone adduct with an ornithine-threonine
dipeptide. Beautifully, even an assignment of the chromophore
absolute configuration emerged from diagnostic distinctions
observed in the spectroscopic properties of the series of all eight
diastereomers derived from the chromophore adduct (four
diastereomers of the two enantiomeric series) that was confirmed
by the total synthesis of the natural product. In retrospect, a
late stage chromophore elaboration, like that of Yao,30 may have
failed to produce sufficient quantities of the natural product for
detection and may not have allowed an unambiguous stereo-
chemical assignment given that the natural product was found
to possess a contrathermodynamic N,O-spiroketal stereochemistry.
The chlorofusin chromophore is derived from a class of
compounds referred to as azaphilones, so named for their ability
to readily condense with ammonia or primary amines.31 In light
of this, at least two potential routes to chlorofusin could be
envisioned: (1) condensation of an appropriate azaphilone with
the full cyclic peptide followed by chromophore oxidation and
spirocycle formation, or (2) construction of the fully function-
alized chromophore appended to a smaller peptide fragment
followed by peptide coupling and macrocyclization to afford
chlorofusin (Scheme 1). The former requires the conduct of an
azaphilone chromophore oxidative spirocyclization on a com-
plex, late stage synthetic intermediate with control of the
resulting relative and absolute stereochemistry or effective
separation and characterization of the resulting diastereomers,
whereas the latter requires that the chromophore N,O-spiroketal
in the smaller peptide fragment not unravel during the late stages
of the ensuing cyclic peptide synthesis. Both routes require the
same precursor azaphilone allowing us to potentially explore
either route as the work progressed.
Because the absolute configuration of the chromophore was
unknown and the relative stereochemical assignment came under
question as the work progressed, the routes pursued in our first
generation synthesis permitted access to both enantiomeric series
and to all possible diastereomers, albeit optimized to access the
original diastereomer assigned by Williams. As discussed herein,
confidence in a requisite reassignment of the relative stereo-
chemistry emerged with the observation of diagnostic spectro-
Results and Discussion
Chromophore Azaphilone Precursor: Route 1. The initial
approach to the key intermediates 17 and 18 used to explore
the C8-C9 oxidative spirocyclization followed established
routestoaccessazaphilonesbearingarangeofC9substituents,32–34
and began with the one carbon homologation of commercially
available 3,5-dimethoxy-4-methylbenzoic acid (5) (Scheme 2).
The crude acid chloride derived from 5 was alkylated with
TMSCH2Bt to afford the ketone adduct (SOCl2, reflux;
(32) Chong, R.; King, R. R.; Whalley, W. B. J. Chem. Soc. C 1971, 3566.
(33) Chong, R.; King, R. R.; Whalley, W. B. J. Chem. Soc. D 1969, 1512.
(34) Suzuki, T.; Okada, C.; Arai, K.; Awaji, A.; Shimizu, T.; Tanemura,
K.; Horaguchi, T. J. Heterocycl. Chem. 2001, 38, 1409.
(31) Powell, A. D. G.; Robertson, A.; Whalley, W. B. Chem. Soc. Spec.
Publ. 1956, No. 5, 27.
9
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A R T I C L E S
Clark et al.
Scheme 2
Scheme 3
on the work of Porco.38 Although a one-step conversion40,41 of
commercially available 2,4-dimethoxy-3-methylbenzaldehyde to
6-bromo-2,4-dimethoxy-3-methylbenzaldehyde suffered from
poor conversions, a more controlled two-step procedure42,43 was
adopted beginning with the condensation of 2,4-dimethoxy-3-
methylbenzaldehyde (19) with N,N′-dimethylethylenediamine
(toluene, 130 °C, 3 h, 95%) (Scheme 3). The resulting
dimethylimidazolidine 20 was regioselectively lithiated (t-BuLi,
Et2O, -55 to 0 °C, 1 h) and subjected to electrophilic
bromination followed by aminal hydrolysis (CF2Br2, -78 to
23 °C, 18 h; 2 N HCl (aq), 30 min, 72%) to provide 6-bromo-
2,4-dimethoxy-3-methylbenzaldehyde (21) in good yield. This
two-step procedure offers more expeditious access to 21 than a
previously reported four-step synthesis.38 Removal of the aryl
methyl ethers proceeded in high yield to give 22 (BBr3, CH2Cl2,
-78 to 23 °C, 88%). Sonogashira coupling of aryl bromide 22
and alkyne 23 provided the azaphilone precursor 24
(Pd(Cl)2(PPh3)2, CuI, Et3N, DMF, 70 °C, 16 h, 73%). Oxidative
cyclization of 24 using Porco’s methodology38 generated the
azaphilone 25 in good yield (Au(OAc)3, TFA, dichloroethane,
4 min; IBX, Bu4NI, 4 h, 72%). Acylation of the tertiary alcohol
of 25 to give 26 (butyric anhydride, DMAP, CH2Cl2, 24 h, 91%),
regioselective C6-chlorination yielding 27 (NCS, AcOH, 36 h,
89%) and removal of the TBDPS protecting group (HF-pyridine,
THF, 2 h, 72%) proceeded smoothly to provide azaphilone 28.
The racemic azaphilones 25, 26, 27 and 28 could be chromato-
graphically separated into their constituent enantiomers, and the
most effective chiral phase resolution was achieved with 27 (R
) 1.33). The absolute stereochemistry of 25-28 was assigned
based on the sign of their optical rotation (RD) and the sign of
the longest wavelength Cotton effect (350-370 nm) in their
CD spectra (Figure 4).44–46 This assignment was further
confirmed by application of Porco’s asymmetric oxidative
TMSCH2Bt, THF, reflux, 6 h)35 and subsequent treatment with
2,6-lutidine and Tf2O provided 6 (2,6-lutidine, Tf2O, CH2Cl2,
0 to 23 °C, 2 h, 86%, three steps). The enol triflate 6 was
converted to the methyl ester 7 by treatment with NaOMe
followed by acidic hydrolysis of the resulting ynamine (NaOMe,
MeCN, reflux, 12 h; 12 N HCl, MeOH, reflux, 24 h, 87%, two
steps). After conversion to Weinreb amide 8 (NHMe(OMe),
i-PrMgCl, THF, -20 °C, 30 min, 95%),36 the lithium reagent
derived from 9 was added to 8 to provide the ketone 10 (9,
t-BuLi, -78 °C, 2 h, Et2O-THF, 90%). Selective deprotection
of the two methyl ethers of 10 in the presence of the silyl ether
using BBr3, BF3 ·Et2O, AlCl3, LiI, TMSI, or PhSH was not
successful. In all cases, the silyl ether was found to preferentially
or nonproductively undergo cleavage. However, the two methyl
ethers were cleaved and the TIPS-protected primary hydroxyl
group was displaced cleanly with iodide to provide 11 in
remarkably high yields when 10 was treated with TMSI under
microwave irradiation (TMSI, MeCN, 120 °C, 50 min, normal
absorption, 95%). Formylation of 11 provided 12 (AlCl3,
CH(OEt)3, toluene, -45 to -15 °C, 1 h, 75%)34 and one-pot
oxidative cyclization of 12 in acetic acid gave the azaphilone
13 while conducting the same reaction in butyric acid provided
14 (p-TsOH, AcOH or butyric acid, 95 °C, 2 h; Pb(OAc)4, 15
°C, 40 min, 48% for 13, 31% for 14).34 Displacement of the
primary iodide of 13 and 14 with acetate (AgOAc, AcOH, 55
°C, 2 h, 87% for 15, 63% for 16)37 gave 15 and 16 whose
regioselective C6-chlorination (NCS, AcOH, 23 °C, 24 h, 87%
for 17, 85% for 18)38,39 afforded the chloroazaphilones 17 and
18.
Chromophore Azaphilone Precursor: Route 2. The analogous
intermediate 28 was also prepared by a more concise route based
(40) Comins, D. L.; Brown, J. D. J. Org. Chem. 1989, 54, 3730.
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U.-H.; Grabowski, E. J. J. Tetrahedron Lett. 1995, 36, 5461.
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(44) Whalley, W. B.; Ferguson, G.; Marsh, W. C.; Restivo, R. J. J. Chem.
Soc., Perkin Trans. 1 1976, 1366.
(38) Zhu, J.; Germain, A. R.; Porco, J. A., Jr. Angew. Chem., Int. Ed. 2004,
43, 1239.
(45) Steyn, P. S.; Vleggaar, R. J. Chem. Soc., Perkin Trans. 1 1976, 204.
(46) Chen, F. C.; Manchand, P. S.; Whalley, W. B. J. Chem. Soc. C 1971,
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A R T I C L E S
23
Figure 4. CD spectra (0.2 mM in MeOH) and RD for azaphilones 25-28 (R ) butyrate).
cyclization47 of the OTIPS variant of 24 conducted using the
catalyst generated from Cu(I) and (-)-sparteine to provide the
corresponding azaphilone (94% ee), matching the assignment
made based on CD. As fate would dictate and since (+)-
sparteine is not yet readily accessible, this asymmetric route
currently only provides access to the chromophore unnatural
enantiomer series.
quickly decomposed and proved challenging to work with or
even purify. In hopes of trapping the labile epoxide, the
analogous azaphilone 29 possessing the unprotected primary
alcohol was treated with DMDO alone or followed with addition
of a catalytic amount of acid (HCl, HOAc, PPTs, CSA).52
Although the formation of the intermediate epoxide was
1
confirmed by H NMR,51 the subsequent spiroketal formation
Oxidative Spiroketalization. With azaphilones 15 and 17 in
hand, we briefly examined their direct oxidative spiroketaliza-
tion. Thus, selective epoxidation or dihydroxylation of the
C8-C9 double bond followed by oxonium ion formation upon
epoxide opening or water elimination was anticipated to install
the C8 alcohol and lead to subsequent C9-spiroketalization with
the pendant side chain alcohol. Confident that the Williams
proposed C8-C9 stereochemistry with the two oxygen substit-
uents syn would be the thermodynamically most stable,
conducting the reaction under conditions of reversible ketal
formation would favor generation of the assigned C8-C9 syn
versus anti diastereomers. Thus, a variety of oxidizing agents
were explored to effect this transformation. Whereas subjecting
azaphilone 15 to the Sharpless asymmetric dihydroxylation
was not observed and the intermediate epoxide was nonpro-
ductively consumed. Consequently, we focused our efforts on
conducting the oxidative spirocyclization following amine
introduction with substrates such as 34 avoiding the issue of
C9-isomerization during amine conjugation.
49
protocol48 or that of oxidative spiroketalization with Re2O7
did not lead to identifiable products, treatment of 15 with
DMDO50 led to epoxidation of the desired C8-C9 double bond
in ca. 40% yield (eq 1). However, the labile epoxide product51
Chromophore Model Studies. We explored a variety of
conditions for condensation of the azaphilones 15 and 17 with
(51) For 30: 1H NMR (500 MHz, CDCl3) δ 7.64 (s, 1H), 6.31 (s, 1H),
5.72 (s, 1H), 4.14 (t, J ) 6.3 Hz, 2H), 2.46 (t, J ) 7.5 Hz, 2H), 2.16
(s, 3H), 2.07 (s, 3H), 1.96 (m, 2H), 1.54 (s, 3H); HR ESI-TOF m/z
351.1072 (M + H+, C17H19O8 requires 351.1074) (Representative
Diastereomer). Treatment of 29 with DMDO followed by concentration
provided a crude 1H NMR indicating the consumption of starting
material and appearance of signals corresponding to epoxide 31 by
analogy to 30.
(47) Zhu, J.; Grigoriadis, N. P.; Lee, J. P.; Porco, J. A., Jr. J. Am. Chem.
Soc. 2005, 127, 9342.
(48) Hashiyama, T.; Morikawa, K.; Sharpless, K. B. J. Org. Chem. 1992,
57, 5067.
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Tetrahedron 2001, 57, 5255.
(50) Adam, W.; Curci, R.; Edwards, J. O. Acc. Chem. Res. 1989, 22, 205.
9
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A R T I C L E S
Clark et al.
Scheme 4
reagents that first activate the C8-C9 double bond by reversible
bromonium or iodonium ion formation.
Thus, early efforts exploring a bromonium ion initiated
spirocyclization, induced upon treatment of 35 with NBS under
anhydrous conditions, provided the R-bromo N,O-spiroketal as
a 1:1 mixture of C4-diastereomers resulting from clean trans
addition across the C8-C9 double bond. Treatment of 35 with
NIS under such conditions failed to provide analogous products
upon workup despite the slow disappearance of starting material
under the reaction conditions. However, inclusion of water in
the reaction mixture led directly to the desired syn R-hydroxy
N,O-spiroketal presumably resulting from water displacement
of the iodide while premature workup of the reaction allowed
isolation of two isomers of the unstable intermediate R-iodo
N,O-spiroketal. Switching from NIS to I2 served to increase the
yield of the syn products and significantly, also led to the
production of trans R-hydroxy N,O-spiroketal products. Al-
though a range of solvents support the reaction (e.g.,
CH3CN-H2O, DMF-H2O), DMSO-H2O provided the syn
cycloadducts in the best yield along with fewer undesired side
products. Finally, the use of Ag(I) salts (AgOTf, AgNO3,
AgClO4, AgOTFA) in the mixture accelerated the reaction,
improved the conversions and minimized a competitive oxida-
tion reaction. Thus, the reaction appears to be initiated by
reversible iodonium ion formation and subsequent iodoetheri-
fication with N,O-spiroketal formation followed by slow Ag(I)-
assisted iodide displacement by H2O providing 36A-36D
directly. Notably, the lone pair of the endocyclic amine is tied
up in the π-system of the chromophore as a vinylogous amide,
and accounts for the relative stability of the iodonium intermedi-
ate, the predominance of C8/C9 syn products arising from SN2-
like trans intramolecular opening of the iodonium ion, and the
high degree of planarity at the nitrogen center observed in the
X-ray crystal structures (Figure 5). The major products (54%),
in which the C8 and C9 oxygen substituents are syn possessing
the C8/C9 stereochemistry found in the Williams assignment,
benzylamine to probe the compatability of the condensation with
solvents that may be required for effective dissolution of the
cyclic peptide or a smaller peptide fragment. Although con-
densation of azaphilone 15 with benzylamine did provide 33 in
modest yield (CH2Cl2, 1-16 h, 54%), condensation of chloro-
azaphilone 17 provided 34 in excellent yield (CH2Cl2, 1 h, 99%)
establishing the productive role of the electron-withdrawing C6-
chlorine substituent (eq 2). Selective hydrolysis of the primary
acetate (K2CO3, H2O, MeOH, 0 °C, 30 min, 75%) followed by
oxidative spirocyclization of 35 with I2 assisted by AgNO3 in
the presence of H2O (I2, AgNO3, DMSO-H2O, 48 h) allowed
access to all four diastereomers (36A-36D) of the simplified
chlorofusin chromophore (Scheme 4).
The development of these conditions followed unsuccessful
efforts to effect a direct oxidative spirocyclization enlisting
DMDO, m-CPBA, peroxyimidate, peroxycarbamate, peroxyi-
sourea, singlet oxygen, TMSOOTMS, as well as Shi, Sharpless,
and Davis asymmetric epoxidation methods or osmium (OsO4),
rhenium (Re2O7), and ruthenium (RuO4) based dihydroxylation
reactions. Hypervalent iodine oxidizing reagents including
Koser’s reagent provided mixtures of 38,53 the oxidized form
of the desired product, and 39,53 representing the allylic
oxidation product (eq 3) although the yields of 38 were
consistently low (10-15%) and the use of alternative reagents
has not yet significantly improved on this. Nonetheless, the
modest success with such reagents initiated studies with related
1
(53) For 38: H NMR (600 MHz, CD3OD) δ 7.94 (s, 1H), 7.38 (m, 5H),
4.83 (d, J ) 15.1 Hz, 1H), 4.75 (d, J ) 15.3 Hz, 1H), 4.21 (dd, J )
14.7, 6.9 Hz, 1H), 4.07 (dd, J ) 14.9, 7.0 Hz, 1H), 2.53 (td, J ) 14.1,
7.1 Hz, 1H), 2.38 (t, J ) 7.2 Hz, 2H), 2.21 (m, 1H), 2.01 (m, 2H),
1.63 (m 2H), 1.53 (s, 3H), 0.97 (t, J ) 7.4 Hz, 3H); 13C NMR
(CD3OD, 150 MHz) δ 190.6, 190.0, 189.4, 172.9, 148.6, 134.9, 134.3,
129.5 (2C), 129.0, 128.2 (2C), 120.4, 102.1, 97.2, 84.5, 71.2, 54.4,
35.2, 33.0, 25.1, 23.1, 18.4, 13.7; IR (film) νmax 2984, 1737, 1374,
1242, 1046, 917, 734 cm-1; HR ESI-TOF m/z 458.1383 (M + H+,
C24H24ClNO6 requires 458.1365) (Representative Diastereomer). For
39: 1H NMR (500 MHz, CDCl3) δ 7.80 (s, 1H), 7.37-7.45 (br m,
3H), 7.19 (d, J ) 7.0 Hz, 2H), 7.10 (s, 1H), 5.26 (d, J ) 16.3 Hz,
1H), 5.10 (d, J ) 16.4 Hz, 1H), 5.00 (dd, J ) 9.1, 4.7 Hz, 1H), 3.82
(m, 1H), 3.75 (m, 1H), 2.44 (dt, J ) 7.3, 3.3 Hz, 2H), 2.28 (m, 1H),
2.20 (m, 1H), 1.67 (m, 2H), 1.56 (s, 3H), 0.97 (t, J ) 7.4 Hz, 3H),
the two OH signals were not observed; 13C NMR (CDCl3, 125 MHz)
δ 193.5, 185.6, 173.3, 148.8, 143.4, 142.4, 134.1, 129.9 (2C), 129.5,
126.6 (2C), 115.1, 114.1, 105.0, 85.0, 77.2, 58.4, 57.1, 39.2, 35.4,
23.1, 18.4, 13.8; MALDI-TOF m/z 460.15 (M + H+, C24H26ClNO6
requires 460.15) (Representative Diastereomer).
(52) Corey, E. J.; Kang, M.; Desai, M. C.; Ghosh, A. K.; Houpis, J. N.
J. Am. Chem. Soc. 1988, 110, 649.
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Total Synthesis of Chlorofusin
A R T I C L E S
Scheme 5
Figure 5. X-Ray structures of 42A and 42D.
S1 and S2, Supporting Information) and bar graph representa-
tions of the chemical shift differences (∆δ, Figures S4 and S5,
Supporting Information) relative to chlorofusin are provided in
the Supporting Information and they, including the diagnostic
distinctions highlighted above, were the first indication that the
C8 and C9 oxygen substituents of chlorofusin may be oriented
anti with respect to one another, and not syn as originally
reported. These may also be summarized more simply as
observed chemical shift differences averaged over the entire
chromophore or restricted to the more diagnostic comparisons
as presented in Scheme 4, both of which indicate that the anti
diastereomer 36D provides the best match with the chlorofusin
chromophore. However, we were concerned about potential
perturbation of the chromophore NMR signals by the appended
benzyl group and elected to examine the additional, more
comparable system 42 incorporating a N-butyl substituent as
well as a C4 butyrate versus acetate. Significantly, the observa-
tions with 36A-36D initiated a switch in our strategy from
targeting the total synthesis of chlorofusin to one that could
also unambiguously establish the chromophore stereochemistry.
As such, the efforts were refocused on preparing and spectro-
scopically characterizing all possible chromophore diastereomers
rather than exclusively targeting the Williams syn isomer.
Thus, butylamine was condensed with chloroazaphilone
18 to give 40 in excellent yield (CH2Cl2, 1 h, 99%).
Hydrolysis of the primary acetate (K2CO3, H2O, MeOH, 0 °C,
30 min, 91%) followed by the single step oxidative spiro-
cyclization of 41 (I2, AgNO3, DMSO-H2O, 48 h) provided
42A-42D possessing the n-butyl substitution (Scheme 5).
As with 36A-36D, the NMR data collected from the anti
isomers 42C and 42D provided a better match with chloro-
fusin than the syn isomers 42A or 42B (Supporting Informa-
tion Tables S3 and S4 and Figures S8 and S9). An X-ray
represent those that formally arise from a trans iodoetherification
reaction followed by water SN2 displacement of the iodide. The
structures of 36A and 36B, the major products of the oxidative
spirocyclization reaction, were established by X-ray crystal-
lography54 and the C8 and C9 oxygen substituents of both
compounds were found to be oriented syn with respect to one
another with the C8-OH occupying an axial orientation. N,O-
Ketal equilibration studies unambiguously established the
structure of the remaining two minor anti diastereomers. The
1H NMR signals of the tetrahydrofuran ring were found to be
diagnostic of the relative orientation of the C8 and C9
substituents. Accordingly, chemical shifts of the C10-H signals
for the two anti isomers 36C (m, 2.25 ppm) and 36D (m, 2.26
ppm) are similar to each other and to the C10-H signal reported
for chlorofusin (br m, 2.38 ppm), but are distinct from the
C10-H signals for 36A (m, 1.89) and 36B (m, 1.81). Similarly
the C11-H signals for 36C (m, 2.01 ppm) and 36D (m, 2.00
ppm) possess chemical shifts that are closer to that of chlorofusin
(m, 2.0-2.2 ppm) than the two syn isomers 36A (m, 1.79 ppm;
m, 1.96 ppm) and 36B (m, 1.76 ppm; m, 1.82 ppm). The
C12-H signals for 36C (m, 3.80 ppm; m, 3.87 ppm) and 36D
(m, 3.77 ppm; m, 3.83 ppm) were also similar to one another
and chlorofusin (q, 3.78 ppm; m, 4.02 ppm), and different from
those of the syn isomers 36A (m, 3.97 ppm; m, 4.18 ppm) and
36B (m, 3.97 ppm; m, 4.18 ppm), but the correlation with the
anti isomers was more tentative. The 13C NMR data exhibited
similar trends, the most striking difference appearing with C10
signals for 36C (30.6 ppm) and 36D (30.5 ppm) being very
similar to each other and to chlorofusin (30.3 ppm), but 4 ppm
farther upfield than the C10 signals for the two syn isomers
36A (34.5 ppm) and 36B (35.0 ppm). A complete tabular
comparison of the spectroscopic properties of 36A-36D (Tables
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A R T I C L E S
Clark et al.
Scheme 6
crystal structure of 42A, one of the major products of the
spirocyclization reaction representing the Williams assigned
diastereomer and the closest match by NMR to 36A,
confirmed that the C4, C8 and C9 oxygen substituents were
all syn while an X-ray of 42D, the closest match with
chlorofusin by NMR, confirmed that the relative orientation
of the C8 and C9 oxygen substituents is anti and that the C4
methyl group is cis to the C8-OH and trans to the C9 oxygen
of the spirotetrahydrofuran (Figure 5).55 N,O-Ketal equilibra-
tion studies established the related syn/anti pairs and
completed the unambiguous stereochemical assignments for
the entire series. As the similarity of the model to the
chlorofusin chromophore increased, the trends in the NMR
data distinguishing the C8/C9 syn diastereomers from the
anti diastereomers became more predominant. For C10-H,
42A (m, 1.85 ppm; m, 1.94 ppm) and 42B (m, 1.82 ppm; m,
2.00 ppm) each display two signals, neither of which is within
0.3 ppm of chlorofusin (br m, 2.38 ppm), whereas the signals
for the two anti isomers 42C (m, 2.37 ppm) and 42D (m,
2.39 ppm) differ from chlorofusin by 0.01 ppm. Similarly,
the C12-H signals for the syn isomers 42A (dd, 4.03 ppm;
m, 4.22 ppm) and 42B (dd, 4.04 ppm; dd, 4.22 ppm) are
much farther downfield than the analogous signals for 42C
(dd, 3.82 ppm; m, 3.95 ppm), 42D (dd, 3.80 ppm; dd, 3.95
ppm) and chlorofusin (q, 3.78 ppm; m, 4.02 ppm). An
additional and important trend emerged linking 42B with
42D, which share the same relative stereochemistry at C8.
(54) Atomic coordinates for the two C8/C9 syn spirocycles 36A
(CCDC646443) and 36B (CCDC646442) have been deposited with
the Cambridge Crystallographic Data Center.
(55) Atomic coordinates for the C8/C9 syn spirocycle 42A (CCDC678265)
and the C8/C9 anti spirocycle 42D (CCDC646441) have been
deposited with the Cambridge Crystallographic Data Center.
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Total Synthesis of Chlorofusin
A R T I C L E S
The signals for C8-H observed in the spectra of 42A (d,
4.48 ppm) and 42C (d, 4.49 ppm) are similar to one another,
whereas the signals for 42B (d, 4.54 ppm) and 42D (d, 4.53
ppm) are not only similar to one another, but also to that of
chlorofusin (d, 4.53 ppm). This, along with other more subtle
differences, distinguishes the two anti diastereomers with
42D, not 42C, being representative of the stereochemistry
found in chlorofusin. Finally, the C8-OH signal in this series
appears to distinguish 42A (d, 6.18 ppm), 42B (d, 6.11 ppm),
42C (d, 6.41 ppm), and 42D (d, 6.25 ppm) as well as link
the relative stereochemistry of 42D with that of chlorofusin
(d, 6.26 ppm).
The 13C NMR show similar trends to the observations made
with 36A-36D. However, the differences between the syn and
anti compounds become even greater in this series for C10, C11,
C12 and C9 in the absence of a proximal phenyl group. With
X-ray structures of representative C8/C9 syn and anti diaster-
eomers in hand, the diagnostic differences seen in their C10
13C NMR chemical shifts can be attributed to its axial (syn) or
equatorial (anti) orientation. For C10, the chemical shifts of 42C
(30.2 ppm) and 42D (30.2 ppm) became even closer to that of
chlorofusin (30.3 ppm), but remained over 4 ppm farther upfield
than the C10 signals for the syn isomers 42A (34.6 ppm) and
42B (35.0 ppm). The butylamine replacement for the Orn side
chain also allowed a comparison of the chemical shift of the
methylene adjacent to the chromophore ring system, with the
anti isomers 42C (50.5 ppm) and 42D (50.6 ppm) matching
that of chlorofusin (50.5 ppm) as opposed to 42A (49.7 ppm)
and 42B (49.8 ppm).
Although intuitively surprising but which now may be
expected from the X-ray crystal structure of 42D (Figure 5)
that shows an unobstructed path between C8-H and C4-Me
(4.761 Å, C8-H to C13), the ROESY NMR for 42D exhibited
a weak cross-peak between C4-Me and C8-H as observed by
Williams as a long-range NOE in chlorofusin. Also observed
in its X-ray structure is the diaxial orientation of the C8 and
C9 oxygen substituents. In this conformation, the equatorial
C8-H can exhibit NOEs to both C10-H and C12-H as
observed by Williams for chlorofusin. Although these NOEs
were not quantitated, it is notable that C8-H is closer to C10-H
(2.484 Å) than C12-H (2.592 Å) in this X-ray consistent with
such implications in the Williams work. In fact, the C8-OH is
axial and the C8-H is equatorial in the X-ray structures for
36A, 36B, 42A, and 42D indicating that the C8-H NOEs
observed by Williams would be possible with all four diaster-
eomers. This dominance of the C8-OH axial orientation arises
presumably from the A(1,3)-strain an equatorial C8-alcohol
would experience with the C6-chloride which is further desta-
bilized by the electronegative nature of the two interacting
substituents (Cl/OH). With this understanding of the confor-
mational properties of the chromophore diastereomers and with
the characterization data from both the 36 and 42 model systems
in hand, the stereochemistry of the chlorofusin chromophore
was confidently reassigned as either the (4S,8R,9S) or (4R,8S,9R)
anti diastereomer (42D enantiomers).
contrathermodynamic product of the cyclization reaction in our
model systems. As such, we elected to take the less ambitious,
more manageable step of linking and elaborating the chro-
mophore on a smaller dipeptide constituting L-Orn9-L-Thr1 of
chlorofusin examining the two enantiomeric series and their four
possible diastereomers. Not only would this provide intermedi-
ates with more manageable physical and spectroscopic properties
and a third model on which to carefully reassess the chlorofusin
stereochemical assignments, but it provided an opportunity to
probe the chromophore absolute stereochemical assignment.
To this end, dipeptide 44 was prepared from the commercially
available amino acid derivatives Fmoc-L-Orn(Mtt)-OH and
L-Thr-OBn using a standard coupling protocol (EDCI, HOAt,
DMF, 24 h, 82%) followed by deprotection of the Nδ-amine of
ornithine (TFA, CH2Cl2, 1 h, 97%), eq 4. Condensation of 44
with either (R)-28 or (S)-28 (NaHCO3, DMF, CH2Cl2, 24 h; 1
N HCl, 4 h, 84% for 45, 71% for 46) provided 45 and 46,
respectively (Scheme 6). While incorporation of simple amines
into the azaphilone core proceeded in high yield with simply
CH2Cl2 as the reaction solvent, condensation with 44 required
more specialized conditions. Solubility considerations led to the
use of DMF as a cosolvent and additive studies revealed that
the early stages of the incorporation were accelerated by addition
of NaHCO3 and subsequent treatment with 1 N HCl (aq) drove
the reaction to completion. Oxidative spirocyclization of 45 (I2,
AgNO3, DMSO-H2O, 72 h) provided 47A-47D and similar
1
treatment of 46 provided 48A-48D. The H and 13C NMR
spectra of all eight diastereomers were fully assigned (Tables
S5-S8, Figures S11-S14, Supporting Information) using
COSY, HMQC, HMBC, and ROESY NMR data and N,O-ketal
equilibration studies defined the related syn/anti pairs in each
enantiomeric series. Notably, the equilibration studies requiring
5% TFA-HOAc (25 °C) revealed that the interconversions, like
those in the 36 and 42 series, are slow even under these strongly
acidic conditions and that the N,O-spiroketals are remarkably
robust. Additionally, resubjecting 47B or 47C to the oxidative
spirocyclization reaction conditions did not provide isomerized
products suggesting that the syn products are both thermody-
namically and kinetically favored and that the anti products
generated are stable to the reaction conditions.
Stereochemical Assignment of Chlorofusin. By using the
diagnostic spectroscopic distinctions observed in the 47 and 48
series, having assigned the C4 absolute stereochemistry by CD
(see Figure 4),44–46 and enlisting N,O-ketal equilibrations to
define the syn/anti pairs in each enantiomeric series, the
complete relative and absolute stereochemical assignments for
all eight diastereomers were unambiguously established. More-
over, only diastereomer 47D (4R,8S,9R) matched all the
spectroscopic properties reported for the chlorofusin chro-
mophore. In short, the two syn and two anti diastereomers in
each enantiomeric series are readily distinguishable by the
diagnostic 1H NMR (C10-H, C11-H, C12-H, and C8-OH)
and 13C NMR (C2 and C9-C12) chemical shifts and supported
by kinetic and thermodynamic predominance of the syn isomers.
L-Orn-L-Thr Azaphilone Conjugation and Elaboration to
All 8 Chromophore Diastereomers. At this stage, we were
convinced that a final stage chromophore coupling to the intact
cyclic peptide followed by oxidative spirocyclization would be
difficult to implement. Not only would this require the separa-
tion, characterization, and unambiguous stereochemical assign-
ments of the four diastereomers generated in each enantiomeric
series, but the reassigned anti isomer represented a minor,
9
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A R T I C L E S
Clark et al.
Figure 8. CD spectra (0.2 mM in MeOH) of 48A-48D.
Scheme 7
Figure 6. Ornithine CH2δ 1H NMR signals for 47A-47D and 48A-48D.
Figure 7. CD spectra (0.2 mM in MeOH) of 47A-47D.
The two anti diastereomers in each enantiomeric series are most
readily distinguished by the diagnostic 1H NMR (C8-H,
C13-H, and C8-OH) and 13C NMR (C3, C7, C8, and C13)
chemical shifts although there are more subtle spectroscopic
distinctions as well. These are discussed in detail in the
Supporting Information where Figures S11-S14 graphically
quantitate these distinctions. In both series, it was the anti
diastereomers 47D and 48D (chromophore enantiomers) that
most closely matched chlorofusin. Additionally, the (4R,8S,9R)-
diastereomer 47D provided a near perfect match with the
spectroscopic properties reported for the chlorofusin chro-
mophore, whereas the (4S,8R,9S)-diastereomer 48D (chro-
mophore enantiomer) proved readily distinguishable by both
given that our examination of the extensive series of known
azaphilones revealed that both C4 configurations are found
naturally with roughly equivalent representations.
As previously discussed, the anti diastereomers adopt a
conformation with both the C8 and C9 oxygen substituents axial
(see Figure 5) and their equatorial C8-H can exhibit NOEs to
both C10-H and C12-H consistent with data for chlorofusin.
The syn diastereomers adopt a conformation in which the
C8-OH is axial, the C9 oxygen substituent is equatorial, and
the C8-H is also equatorial. These isomers similarly exhibit
C8-H to C10-H and C12-H NOEs. However, an important
1
the H NMR chemical shift and multiplicity of the ornithine
δ
CH2 adjacent to the chromophore (δ 3.45, m, 2H for 47D vs
δ 3.41 and 3.52, two m, 1H each for 48D; chlorofusin ) δ
3.42, t, 2H). This final multiplicity distinction between 47D and
48D allowed the absolute stereochemical assignment for the
chlorofusin chromophore (Figure 6). This assignment of the
absolute configuration for chlorofusin is especially significant
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Total Synthesis of Chlorofusin
A R T I C L E S
Scheme 8
Scheme 9
ramification of the conformational features of the syn and anti
diastereomers where each possess an equatorial C8-H is the
counterintuitive observation that all four diastereomers possess
nearly equivalent C4-Me to C8-H distances (5.42-5.55 Å)
and that it is actually closest (5.42 Å) for the anti isomer we
assigned to chlorofusin. The ROESY NMR data (300 ms mixing
time) for these eight diastereomers reveals an NOE only between
C8-H and C4-Me (and not C4-Me/C8-OH) when those
groups are cis with respect to one another (47A, 47C, 48A,
and 48C) and a correlation between C4-Me and both C8-H
and C8-OH when C4-Me is trans with respect to C8-H (47B,
47D, 48B, and 48D). Thus, regardless of the relative stereo-
chemistry of the chromophore, a NOE is seen between C4-Me
and C8-H making the reassignment of the chromophore relative
stereochemistry consistent with Williams’ spectroscopic char-
acterization of the natural product. Importantly, the NOE data
collected and reported by Williams is impeccable including that
of the long-range C4-Me/C8-H NOE, but it did not prove
diagnostic of a C4-Me/C8-H cis stereochemistry.
positive longest wavelength Cotton effect is diagnostic of the
C4-R stereochemistry and a negative Cotton effect is diagnostic
of the C4-S stereochemistry.44–46
Total Synthesis of Chlorofusin and its Three 4R-Diastereo-
mers. With the chromophore stereochemistry confidently as-
signed, the (4R,8S,9R)-diastereomer 47D was incorporated into
Comparison of the CD spectra of all eight diastereomers
shows that the region between 250-500 nm is dependent solely
on the stereochemistry of the chromophore with nearly equal
and opposite spectra observed for 47A and 48A, for 47B and
48B, for 47C and 48C, and for 47D and 48D (Figures 7 and
8). Of particular note is the sign of the longest wavelength
Cotton effect (395-410 nm).56 As with azaphilones 25-28, a
Figure 9. CD spectra (0.2 mM in MeOH) of 3, 65, 66, and 1.
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A R T I C L E S
Clark et al.
coupling and macrocyclization reaction conditions and their
workup. Extended treatment (16 h) with H2/Pd-C does slowly
hydrogenate the material, but it does not isomerize it. We
minimized this potentially competitive reaction during the benzyl
ester and Cbz deprotections using short reaction times (4 h).
The Fmoc deprotection with piperidine (40 min) proceeded
uneventfully providing the free amines of 47A-47D, each of
which was isolated by chromatography, without detection of
isomerization or other conceivable problems. As such, the
stability of the N,O-ketal is greater than one might intuitively
suspect and this may be attributed to the fact that the nitrogen
is tied up as a vinylogous amide. Two empirical observations
additionally highlight this. First, the isolation of chlorofusin was
conducted with TFA (0.01%) in the reverse phase HPLC solvent
system.23 Second, the conditions required for N,O-ketal isomer-
ization are strongly acidic (e.g., 5% TFA-HOAc or 1:1
TFA-CH2Cl2, 5 h) and they do not isomerize under the
conditions of a mild aqueous acid workup. Thus, a projected
concern over the chromophore stability did not materialize,
simplifying the path forward.
Figure 10. CD spectra of synthetic versus natural chlorofusin.
a total synthesis of chlorofusin. As a prelude to our initial
assemblage of chlorofusin, one of the two anti diastereomers
(47C) in the natural 4R-enantiomeric series was exposed to
many of the reaction conditions and recovered unchanged. Its
treatment with EDCI-HOAt and NaHCO3 in DMF for 16 h
followed by exposure to workup conditions (dilute with EtOAc,
wash with 1 N HCl (2×), aq. NaHCO3 (2×), saturated aq. NaCl,
and drying over Na2SO4) provided a quantitative recovery of
47C with no detectable isomerization to its more stable syn
isomer (47A). This would be representative of the peptide
Peptide 61, synthesized in a convergent sequence (Scheme
7), was protected in a manner that avoids treatment of late stage
chromophore-containing intermediates with strong acid that
might promote C9 N,O-spiroketal isomerization. Fmoc depro-
tection of 47D (piperidine, DMF-CH2Cl2, 40 min) and coupling
of the free amine 62 with heptapeptide 61 (EDCI, HOAt,
Figure 11. 1H NMR overlay of natural chlorofusin with all four C4-R chlorofusin chromophore diastereomers.
9
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Total Synthesis of Chlorofusin
A R T I C L E S
Scheme 10
Figure 12. CD spectra (0.2 mM in MeOH) of 2, 67, 68, and 4.
deprotection of the chromophore dipeptide precursor and EDCI-
promoted coupling with the same heptapeptide 61, concurrent
Cbz and benzyl ester deprotection, and a final macrolactam-
ization to provide diastereomers 3, 65, and 66 with purification
of a single intermediate in addition to the final products (Scheme
9). This late stage divergence in a convergent total synthesis
facilitated the assemblage of the collective set of diastereomers
and the anticipated noncorrelation of their spectroscopic proper-
ties with that reported for the natural product provided further
firm support for the new structural assignment. The spectro-
scopic distinctions in these isomers mirrored those observed with
the dipeptide conjugates 47A-47D with the two syn and two
anti diastereomers in each enantiomeric series readily distin-
guishable by the diagnostic 1H NMR (C10-H, C11-H,
C12-H, and C8-OH) and 13C NMR (C2 and C9-C12)
chemical shifts. The two anti diastereomers in each enantiomeric
series are most readily distinguished by the diagnostic 1H NMR
(C8-H, C13-H, and C8-OH) and 13C NMR (C3, C7, C8,
and C13) chemical shifts along with more subtle spectroscopic
distinctions.
Following the completion of this work and our chlorofusin
structural reassignment and upon the Yao report30 that claimed
to have prepared chlorofusin (4S,8R,9S-diastereomer), we re-
examined a sample of authentic chlorofusin provided by Dr.
Stephen Wrigley in 2003 (aged and of unknown quality) that
failed to provide a discernible 1H NMR spectrum in 2003. Now,
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 the absolute configuration assignment
(Figure 10). Moreover, its CD spectrum was readily distinguish-
able from the two possible syn diastereomers (4R,8R,9R and
4R,8S,9S) and more subtly distinguishable from the alternative
anti (4R,8R,9S)-diastereomer 66 (Figure 9). Like the simple
azaphilone chromophore precursors 25-28, the dipeptide aza-
philone conjugates 45 and 46, as well as the fully elaborated
chromophore dipeptides 47 and 48, the longest wavelength
(395-415 nm) CD Cotton effect is positive for all four 4R
chlorofusin diastereomers as well as the natural product firmly
establishing the absolute configuration assignment.44–46
Just as significantly, the sample provided an 1H NMR
spectrum of a sufficient quality to confirm that it represented
the authentic natural product (Supporting Information). Il-
lustrated in Figure 11 is the spectral overlay of a diagnostic
region of the 1H NMR spectra of this sample of natural
NaHCO3, DMF, 0 to 23 °C, 24 h, 55%) provided 63 (Scheme
8). Concurrent benzyl ester deprotection and Cbz removal (H2,
Pd-C, THF-DMF, 4 h) followed by macrocyclization of 64
(EDCI, HOAt, NaHCO3, DMF, 0 to 23 °C, 40 h, 60%) afforded
synthetic chlorofusin (1), which displayed spectroscopic proper-
ties indistinguishable from those reported for natural chlorofusin.
Among the most notable of these spectroscopic correlations was
the single signal for the ornithine CH2δ (δ 3.42, t, 2H for natural
and synthetic chlorofusin) that readily distinguishes the natural
diastereomer from the (4S,8R,9S)-diastereomer incorporating the
chromophore enantiomer (δ 3.42 and 3.50, two m, 1H each)
that was prepared and miscorrelated by Yao.30
In addition to the natural (4R,8S,9R)-diastereomer constituting
chlorofusin, the (4R,8R,9R)-diastereomer 3 proposed by Wil-
liams as well as the (4R,8S,9S)- and (4R,8R,9S)-diastereomers
of chlorofusin (65 and 66) were also prepared by this route from
47A-47C. Notably, this entailed simply the four steps of Fmoc
(56) There is an interesting shift in the longest wavelength CD Cotton effect
from 360 nm for the azaphilones 25-28 (Figure 4), to 375-380 nm
for the (dipeptide) amine conjugates 45 and 46, to 395-410 nm for
47A-47D and 48A-48D.
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A R T I C L E S
Clark et al.
Figure 13. 1H NMR overlay of natural chlorofusin with all four C4-S chlorofusin chromophore diastereomers.
chlorofusin with the four 4R diastereomers illustrating the clear
correlation with only the synthetic (4R,8S,9R)-diastereomer 1
and the clear distinctions with any of the alternative 4R
diastereomers in this enantiomeric series. Significantly, the syn
diastereomer 3 proposed by Williams is readily discernible from
displayed an anticipated longest wavelength negative Cotton
effect opposite that observed with the natural product (Figure
12). Moreover, each 4S-diastereomer provided an essentially
identical, but of opposite sign, CD spectrum (250-500 nm) to
the corresponding 4R-diastereomer indicating that it is the
chromophore, and not the cyclic peptide, that establishes the
sign and magnitude of the CD spectrum in this region. Notably,
that of 4 which bears the chlorofusin chromophore enantiomer
is nearly identical to that of chlorofusin, but is clearly of the
opposite sign.
1
natural chlorofusin not only by CD (Figure 9), but also by H
NMR (Figure 11). Additionally, the comparisons in this region
of the 1H NMR spectra clearly confirm the stereochemical purity
and integrity of each isomer indicating that each, including the
less stable anti diastereomers, is unaffected by the synthetic
sequence required for their incorporation into the final products.
Synthesis of All Four 4S-Diastereomers of Chlorofusin. With
the Yao disclosure of the synthesis and miscorrelation of 4 with
chlorofusin,30 we elected to extend our studies to include the
preparation and characterization of all four 4S-chromophore
diastereomers of chlorofusin including 4. The preparation of
this enantiomeric series simply required the late-stage individual
incorporation of 48A-48D into the full chlorofusin structure
and again benefited from the convergent nature of the total
synthesis providing diastereomers 2, 67, 68, and 4 in four steps
of which 4 corresponds to the Yao structure (Scheme 10).
Though operationally short, the final materials are technically
challenging to handle (SiO2 and C18-reverse phase chromatog-
raphy) making their synthesis more challenging than our
expression of access in four steps might inadvertently convey.
With 2, 67, 68, and 4 in hand, their spectroscopic properties
proved readily distinguishable from the natural product. Each
Similarly, the 1H NMR and 13C NMR spectroscopic proper-
ties of each diastereomer 2, 67, 68, and 4 were distinguishable
from those of chlorofusin (see Supporting Information Table
S11 and S12). This is most apparent in the region represented
with the spectral overlay with the natural product in Figure 13.
The Yao diastereomer 4, bearing the enantiomer of the chro-
mophore found in natural chlorofusin, is most readily distin-
δ
guishable by its ornithine CH2 signal which appears as two
multiplets of 1H each (δ 3.41 and 3.52), but also exhibits more
subtle differences including C8-OH (δ 6.22 vs 6.26), Orn9
R-CH (δ 4.56 vs 4.59), Leu5 R-CH (δ 4.45 vs 4.48), Thr1 R-CH
(δ 3.68 vs 3.66), Asn3 ꢀ-CH2 (δ 2.57, dd vs 2.62, app t),
C10-H (δ 2.40, m vs 2.38, br m), and C11-H (δ 2.04, m vs
2.0-2.2, br m). Similarly, the alternative syn diastereomer 2
proposed by Williams and in this enantiomeric series also
exhibited spectroscopic properties readily distinguishable from
δ
the natural product. Interestingly, while the ornithine CH2
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12368 J. AM. CHEM. SOC. VOL. 130, NO. 37, 2008

Total Synthesis of Chlorofusin
A R T I C L E S
signals for 1, 65, 66, and 3 follow the trend observed with
47A-47D appearing as a single multiplet of 2H in the 1H NMR
when the stereochemistry at C8 is S and as two multiplets of
1H each when the stereochemistry at C8 is R, it was observed
that the ornithine CH2δ signal for 2, 67, 68, and 4 appeared as
distinguishable multiplets of 1H each independent of the C8
stereochemistry.
readily distinguishable from the natural product. This included
the diastereomer advanced by Yao30 as being identical with
spectroscopic properties reported for natural chlorofusin as well
as both enantiomeric chromophores of the original Williams
assignment.23 This study with the comprehensive characteriza-
tion of all possible isomers was accomplished enlisting a
convergent synthetic strategy with a key late-stage divergent
introduction of the eight chromophore-dipeptide conjugates
requiring only four steps for completion of the synthesis and
facilitating the total synthesis of the full series of eight
chromophore diastereomers. Currently, efforts are underway to
synthesize the chromophore stereoselectively as well as to probe
the mechanism by which chlorofusin disrupts the interaction
of p53 and MDM2.
Conclusion
Full details of the first total synthesis of chlorofusin are
reported resulting in the reassignment of its chromophore relative
stereochemistry and the establishment of its chromophore
absolute stereochemistry. Careful spectroscopic characterization
of each of the four chlorofusin model chromophore diastereo-
mers in two distinct series suggested the initial Williams
assignment might not prove accurate and revealed that the key
spectroscopic feature leading to the original assignment does
not distinguish the four possible diastereomers. Subsequent
preparation and characterization of all four diastereomers of a
dipeptide conjugate with both enantiomers of the chlorofusin
chromophore confirmed the required relative stereochemistry
reassignment and permitted an assignment of its absolute
configuration. This was unambiguously established with the total
synthesis of not only chlorofusin, which displayed spectroscopic
and physical properties indistinguishable from the natural
product, but also all seven alternative chlorofusin chromophore
diastereomers, each of which displayed spectroscopic properties
Acknowledgment. Dedicated to Professor E. J. Corey on the
occasion of his 80th birthday. We gratefully acknowledge the
financial support of the National Institutes of Health (CA41101)
and the Skaggs Institute for Chemical Biology. We are especially
grateful to Dr. S. S. Pfeiffer for initial studies on the chromophore
synthesis (Scheme 2) and both Dr. S. S. Pfeiffer and Dr. P. Desai
for initial studies on the chlorofusin cyclic peptide.26
Supporting Information Available: Full experimental details
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA8012819
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