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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Prod Rep. Author manuscript; available in PMC 2010 April 1.
Published in final edited form as:
Nat Prod Rep. 2009 April; 26(4): 465–477.
PMCID: PMC2719831

The isolation, total synthesis and structure elucidation of chlorofusin, a natural product inhibitor of the p53-MDM2 protein-protein interaction


Inhibitors of key protein-protein interactions are emerging as exciting therapeutic targets for the treatment of cancer. One such interaction between MDM2 (HDM2) and p53, that silences the tumour suppression activities of p53, was found to be inhibited by the recently isolated natural product chlorofusin. Synthetic studies on this complex natural product summarized herein have served to reassign its chromophore relative stereochemistry, assign its absolute stereochemistry, and provided access to a series of key analogues and partial structures for biological evaluation.

1 Introduction

1.1 p53-MDM2

The tumor suppressor p53 is an important part of an innate protection mechanism where it acts as a transcription factor that initiates cell cycle arrest and apoptosis in response to DNA damage.1-4 The activity of p53 is modulated by MDM2 (HDM2), which 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 implicated in many cancers,10-16 defining the p53-MDM2 interaction as an attractive target for therapeutic intervention.4,17 An X-ray crystal structure of the N-terminal domain of MDM2 bound to the 15-residue transactivation domain of p53 revealed that the formation of the complex is mediated by the interaction of three hydrophobic residues of a p53 α-helix with a hydrophobic cleft of MDM2.18 Molecules that bind this hydrophobic cleft of MDM2 disrupt the protein-protein interaction with p53, restoring the regulatory function of p53 and inhibiting tumor growth.4,19-22

1.2 Isolation, initial structural assignment, and activity of chlorofusin

In 2001, chlorofusin (1, Fig. 1) was reported by Williams as the most abundant MDM2-p53 inhibitor isolated from the fermentation broth of a Fusarium species of microfungus (corrected as Microdochium caespitosum) in the course of an activity-guided screening program involving the examination of over 53,000 microbial extracts.23 Chlorofusin was found to disrupt the MDM2-p53 interaction in a DELFIA-modified ELISA assay (IC50 = 4.6 μM), but was inactive against the TNFα:TNFα protein-protein interaction and showed no cytotoxicity against Hep G2 cells at 4 μM. Through the use of surface plasmon resonance (SPR) spectroscopy, chlorofusin was later shown to directly bind the N-terminal domain of MDM2 (KD = 4.7 μM) and fitting of a biphasic model to the data has been interpreted as an initial fast binding event followed by a second slow step.24 Thus, chlorofusin represents an exciting lead for antineoplastic intervention that acts by a rare disruption of a protein-protein25 interaction although the structural details of its interaction with MDM2 have yet to be established.

Fig. 1

On the basis of thorough spectroscopic and chemical 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 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. These studies provided the cyclic peptide structure and connectivity, although the stereochemical assignment of the two asparagine residues (Asn3 and Asn4) could not be made and were simply established to have opposite stereochemistries (L and D).

Similarly, the structure and relative stereochemistry of the unusual azaphilone-derived chromophore was also proposed, although an assignment of its absolute stereochemistry was not possible (Fig. 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 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 were interpreted to indicate that the protons attached to C8, C10 and C4-Me all lie on the same face of the chromophore and led to the initial relative stereochemical assignment represented by 2 and 3 in Fig. 2.

Fig. 2
Original chromophore relative stereochemical assignment. No assignment of absolute stereochemistry was made.

1.3 Biosynthesis of chlorofusin

To date, a single investigation aimed at defining the biosynthesis of chlorofusin has been conducted by growing Microdochium caespitosum in a 13C-labeled acetate (CH313CO2Na) growth medium and examining the isolated natural product using 13C NMR spectroscopy.26 Strong 13C NMR signal enhancement was observed for carbons 1, 3, 5, 7, 9, 11, 15, 17, alternating carbons along the alkyl chain of the 2-aminodecanoic acid residue, and the carbonyls of Leu5, Leu7 and ADA8 (Fig. 3). The backbone and side chain carbonyls of the two asparagine residues were also enriched to a lesser degree with 13C. These results are consistent with the biosynthetic origin of common amino acids27 and serve to establish the acetogenic origin of the 2-aminodecanoic acid residue. The alternating incorporation of 13C into the chromophore establishes the acetogenic origin of this structure and is in agreement with previous studies on the fungal biosynthesis of the azaphilones.28

Fig. 3
Chlorofusin carbons with enhanced 13C NMR signals derived from 13C labelled acetate.

2 Cyclic peptide syntheses

2.1 Boger cyclic peptide synthesis

In 2003, the Boger group reported the synthesis of two chlorofusin cyclic peptide diastereomers bearing either the l-Asn3/d-Asn4 or d-Asn3/l-Asn4 stereochemistry and correlated the former with the spectroscopic properties (1H and 13C NMR) of the natural product.29a In this work, three key subunits were assembled, sequentially coupled, and cyclized to provide the 27-membered nine residue cyclic peptide (Scheme 1). 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.

Scheme 1
Boger solution phase synthesis of the chlorofusin cyclic peptide.29a

The data obtained from COSY, HMBC, and HMQC NMR experiments indicated that the 1H and 13C NMR chemical shifts of peptide 17 closely matched those reported for the natural product as compared to those of peptide 15. Characteristic of this similarity in 1H NMR chemical shifts, the nine α-CH chemical shifts of 17 exhibited ≤ 0.02 δ differences from those reported for chlorofusin (av Δδ = 0.016), whereas those of 15 exhibited much larger differences of 0.1-0.6 δ (av Δδ = 0.34). Analogous comparisons of the 13C NMR shifts of the α-carbons provide similar distinctions (15, Δδ = 0.5-3.0, av Δδ = 1.91 vs 17, Δδ = 0.0-0.8, av Δδ = 0.47). In addition, Williams reported a series of diagnostic long range NOEs for the cyclic peptide, including a key NOE between both β-methylene hydrogens of Asn3 and the two methines of Thr6 including the α-H (C26-H). While not as extensively defined as those in the Williams study, the long range NOEs observed in the NOESY and ROESY NMR experiments for 15 and 17 were sufficient to indicate that peptide 17 satisfies the previously reported NOEs while 15 does not.

Removal of the Orn9 side chain SES protecting group of 15 and 17 afforded the corresponding free amines and permitted the synthesis of a series of Orn9 derivatives (e.g., NHCbz, NHFmoc, NHBoc, N-phthalimide, and NH-naphthalene acetamide). Cyclic peptides 15 and 17, and their corresponding free amines, along with their Orn9 derivatives were assayed for inhibition of MDM2/p53 binding in an ELISA assay enlisting p53 immobilized via a biotin linker and full length MDM2, however no activity was observed (IC50 > 250 μM).

More recently, Boger and Lee29b have reported an alternative and improved convergent synthesis of 17 enlisting the same three peptide subunits assembled in an alternative order with the final macrocyclization conducted at the more favorable d-ADA/l-Orn site.

2.2 Searcey cyclic peptide synthesis

A disclosure by Searcey and coworkers concurrent with that of Boger described the synthesis of the l-Asn3/d-Asn4 diastereomers of the chlorofusin cyclic peptide incorporating either a d-ADA8 (18) or l-ADA8 (19) residue and served to verify the assigned d-stereochemistry of the 2-aminodecanoic acid residue.30 In this work, the linear peptides were efficiently constructed and cyclized on solid phase utilizing an l-Asp residue immobilized on a Rink amide resin (Scheme 2). Initially the ADA8 was installed as a racemic mixture and the final compounds were separated by HPLC, and a subsequent resynthesis utilizing enantiomerically pure d-Fmoc-ADA-OH provided 19 and allowed the stereochemical assignment of each product. Highlights of this strategy include concurrent side chain deprotection/product release from the resin and the requirement for only one late stage HPLC purification step.

Scheme 2
Searcey solid phase synthesis of the chlorofusin cyclic peptide.

Most of the NOEs observed for chlorofusin23 were detected in the NOESY spectrum of 19, including the cross peaks between Leu5 and ADA8 and between Asn3 and Thr6, although the presence of additional cross peaks suggested conformational differences from the chromophore-bearing natural product. In contrast, the NOESY spectrum of 18 had notable differences when compared with that for chlorofusin, including the absence of a long range cross peak between Asn3 and Thr6. The diastereomer 19 incorporating the d-ADA residue was found to be a close match to the data reported for chlorofusin, 18 exhibited substantial spectroscopic differences. Screening of these two compounds in an osteosarcoma cell line which contains wild type p53 and overexpresses HDM2, the human homologue of MDM2, provided no discernable evidence of p53 activation.30

In an extension of their initial work and demonstrating the utility of their solid phase synthesis, several analogues of the chlorofusin cyclic peptide were prepared in a subsequent study by Searcey31 (Fig. 4). Based on an examination of cyclodecapeptide β-hairpin constructs with high binding affinities for MDM2,32 they proposed that Ala2, Orn9, and d-ADA8 of the chlorofusin peptide may be responsible for its interaction with the p53 binding site on MDM2. Thus, the peptide analogues explored contained modifications at the Ala2 and Orn9 positions. A key element of this approach is the on-resin cyclisation that allows analogue synthesis while still immobilized on the solid phase. Orthogonal use of an Mtt (methyltrityl-) protecting group on the Orn9 side chain amine led to the selective on-resin deprotection (using 1% TFA) of Orn9-δNH2 that was then reacted with various acid chlorides. The resultant substituted benzamides were chosen to mimic possible interactions involving the 4-, 5-, 6-, and 7-positions of the chromophore with MDM2. As in their previous work, this strategy required only one final global deprotection/cleavage step and only one HPLC purification step for each analogue constructed.

Fig. 4
Searcey chlorofusin cyclic peptide analogues.

Evaluation of these eight cyclic peptide analogues was conducted in an ELISA assay adapted from the work of Kahne33 and measured disruption of the interaction between HDM2 (17-125) and immobilized biotinylated SGSG-p53 peptide (17-27). Although the control p53 peptide (SQETFSDLWKLLPEN) produced an IC50 value of 4.2 μM in this assay, comparing well with reported values, the chlorofusin analogues displayed no p53/MDM2 inhibitory activity with IC50 values outside their solubility range in the assay.

2.3 Nakata cyclic peptide syntheses

Recently, Nakata34 has reported an additional solution phase synthesis of the chlorofusin cyclic peptide employing a Boc/TMSE strategy and key macrocyclization at the d-ADA/l-Orn site, which features the late stage incorporation of the l-Orn9 residue in order to expedite and anticipate the synthesis of chlorofusin and its chromophore analogues.

3 Boger total synthesis, structural reassignment, and absolute configuration of chlorofusin

3.1 Retrosynthetic analysis

The chlorofusin chromophore is derived from a class of compounds referred to as azaphilones, so named for their ability to condense with ammonia or primary amines.35 Given what is known about the electrophilic nature of the azaphilones, at least two potential routes to chlorofusin could be envisioned: (1) condensation of an appropriate azaphilone with the intact cyclic peptide followed by chromophore oxidative spirocyclization, or (2) construction of the fully functionalized chromophore appended to a smaller peptide fragment followed by peptide coupling and macrocyclization to afford chlorofusin. The former requires the conduct of an azaphilone chromophore oxidative spirocyclization on a complex, 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.

Since the absolute configuration of the chromophore was unknown and the relative stereochemical assignment came under question as the work progressed, the route pursued in the Boger groups first generation synthesis36,37 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 stereochemistry emerged with the observation of diagnostic spectroscopic distinctions (1H and 13C NMR) made possible by access to all diastereomers. Due to concerns for an unambiguous stereochemical assignment as the studies progressed, the strategy of early stage chromophore oxidative spirocyclization with clear stereochemical assignments for all diastereomers became the focus of these efforts and was effectively conducted on the azaphilone adduct with an ornithine-threonine dipeptide. Ultimately, 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 in each of the two enantiomeric series) that was ultimately confirmed by the total synthesis of the natural product. In retrospect, a late stage chromophore elaboration may have failed to produce sufficient quantities of the natural product for detection and may not have allowed an unambiguous stereochemical assignment given that the natural product was found to possess a contrathermodynamic N,O-spiroketal stereochemistry.

3.2 Chlorofusin chromophore synthesis

The azaphilone intermediate 29 was initially prepared by a route based on the work of Whalley,38 and subsequently accomplished using a second, more concise route based on the work of Porco,39 the latter of which is shown in Scheme 3. Regioselective bromination of 20, via o-lithiation of 21, was followed by demethylation under standard conditions (BBr3, 88%) to generate 23. Sonogashira coupling of aryl bromide 23 and alkyne 24 provided the azaphilone precursor 25 (Pd(Cl)2(PPh3)2, CuI, Et3N, 73%). Oxidative cyclization of 25 using Porco’s methodology39 generated the azaphilone 26 in good yield (Au(OAc)3, CF3COOH, dichloroethane, 72%). Acylation of the tertiary alcohol of 26 to give 27 (butyric anhydride, 91%), regioselective C6-chlorination yielding 28 (NCS, 89%) and removal of the TBDPS protecting group (HF-pyridine, 72%) proceeded smoothly to provide azaphilone 29. The racemic azaphilones 26-29 could be chromatographically separated into their constituent enantiomers, and the most effective chiral phase resolution was achieved with 28 (α = 1.33). The absolute stereochemistry of the enantiomerically pure azaphilones was assigned based on the sign of their optical rotation (αD) and the sign of the longest wavelength Cotton effect (350-370 nm) in their CD spectra (Fig. 5).40-42 This assignment was further confirmed by application of Porco’s asymmetric oxidative cyclization43 of the OTIPS variant of 25 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.

Fig. 5
CD spectra (0.2 mM in MeOH) and αD23 for azaphilones 26 and 29 (R = butyrate).
Scheme 3
Boger second generation azaphilone synthesis.

3.3 Chromophore model studies

Following extensive exploration of direct and indirect oxidative spirocyclization methods,39 oxidative spirocyclization with I2 assisted by AgNO3 in the presence of DMSO-H2O (I2, AgNO3, H2O, DMSO, 48 h) allowed access to all four diastereomers (33A-33D) of a simplified chlorofusin chromophore (Fig. 6).

Fig. 6
Model chromophore diastereomers 33A-33D and 37A-37D. Tables show the average variation in total and diagnostic chemical shifts in proton and carbon spectra when compared with Williams’ original data.23

This reaction appears to be initiated by reversible iodonium ion formation and subsequent iodoetherification with N,O-spiroketal formation followed by slow Ag(I)-assisted iodide displacement by H2O providing the desired products 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 intermediate, and the predominance of C8/C9 syn products arising from SN2-like trans intramolecular opening of the iodonium ion. The major products (54%), in which the C8 and C9 oxygen substituents are syn possessing the C8/C9 stereochemistry found in the original Williams assignment, represent those that formally arise from a trans iodoetherification reaction followed by water SN2 displacement of the iodide. The structures of 33A and 33B, the major products of the oxidative spirocyclization reaction, were established by X-ray crystallography 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. A detailed comparison of the spectroscopic properties of 33A-33D to chlorofusin provided 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 assigned. For the purposes herein, these may be summarized simply as observed chemical shift differences averaged over the entire chromophore or restricted to the more diagnostic comparisons as presented in Fig. 6, both of which indicate that the anti diastereomer 33D provides the best match with the chlorofusin chromophore. However, the chromophore NMR signals could be impacted by the appended benzyl group and an additional, more comparable system 37 was examined, incorporating an N-butyl substituent as well as a C4 butyrate versus acetate. Significantly, the observations with 33A-33D initiated a switch in the strategy from targeting the total synthesis of chlorofusin by this group to one that could also unambiguously establish the chromophore stereochemistry. As such, the efforts were refocused on preparing and spectroscopically characterizing all possible chromophore diastereomers rather than exclusively targeting the Williams syn isomer.

Thus, chromophore derivatives 37A-37D possessing the N-butyl substitution were synthesized (Fig. 6). As with 33A-33D, the NMR data collected from the anti isomers 37C and 37D provided a better match with chlorofusin than the syn isomers 37A or 37B. An X-ray crystal structure of 37A, representing the Williams-assigned diastereomer and the closest match by NMR to 33A, confirmed that the C4, C8 and C9 oxygen substituents were all syn, whereas an X-ray of 37D, 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 (Fig. 7). N,O-Ketal equilibration 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. This, along with other more subtle differences, distinguished the two anti diastereomers with 37D, not 37C, being representative of the stereochemistry found in chlorofusin.

X-Ray structures of 37A and 37D.

Although intuitively surprising but which now may be expected from the X-ray crystal structure of 37D (Fig. 7) that shows an unobstructed path between C8-H and C4-Me (4.761 Å, C8-H-C13), the ROESY NMR for 37D 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 33A, 33B, 37A and 37D indicating that the C8-H NOEs observed by Williams would be possible with all four diastereomers. This dominance of the C8-OH axial orientation arises from the A(1,3)-strain an equatorial C8-alcohol would experience with the C6-chloride that is further destabilized by the electronegative nature of the two interacting substituents (Cl/OH). With this understanding of the conformational properties of the chromophore diastereomers and with the characterization data from both the 33 and 37 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 (37D enantiomers).

3.4 l-Orn-l-Thr azaphilone conjugation and elaboration to all eight chromophore diastereomers and stereochemical assignment of chlorofusin

At this stage, it was clear 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 separation, characterization, and unambiguous stereochemical assignment of the four diastereomers generated in each enantiomeric series, but the reassigned anti isomer represented a minor, contra-thermodynamic product of the cyclization reaction in the model systems. As such, the less ambitious, more manageable step of linking and elaborating the chromophore on a smaller dipeptide constituting l-Orn9-l-Thr1 of chlorofusin was examined using both enantiomeric series and their four possible diastereomers. Not only did this provide intermediates 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 38 was condensed with either (R)-29 or (S)-29 (84% for 39, 71% for 41) to provide 39 and 41, respectively (Fig. 8). Oxidative spirocyclization of 39 (I2, AgNO3, H2O, DMSO, 72 h) provided 40A-40D and similar treatment of 41 provided 42A-42D. The 1H and 13C NMR spectra of all eight diastereomers were fully assigned 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 33 and 37 series, are slow even under these strongly acidic conditions and that the N,O-spiroketals are remarkably robust. Additionally, resubjecting 40B or 40C to the oxidative spirocyclization reaction conditions did not provide isomerized products suggesting that the syn products are both thermodynamically and kinetically favored and that the anti products generated are stable to the reaction conditions.

Fig. 8
Synthesis of all eight l-Orn9-l-Thr1 conjugated chromophore diasteromers. Tables show the average variation in total and diagnostic chemical shifts in proton and carbon spectra when compared with Williams’ original data.23

By using the diagnostic spectroscopic distinctions observed in the 40 and 42 series, having assigned the C4 absolute stereochemistry by CD (see Fig. 5),40-42 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. Moreover, only diastereomer 40D (4R,8S,9R) matched all the spectroscopic properties reported for the chlorofusin chromophore. 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. 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. In both series, it was the anti diastereomers 40D and 42D (chromophore enantiomers) that most closely matched chlorofusin. Additionally, the (4R,8S,9R)-diastereomer 40D provided a near perfect match with the spectroscopic properties reported for the chlorofusin chromophore, whereas the (4S,8R,9S)-diastereomer 42D (chromophore enantiomer) proved readily distinguishable by both the 1H NMR chemical shift and multiplicity of the ornithine CH2δ adjacent to the chromophore (δ 3.45, m, 2H for 40D vs δ 3.41 and 3.52, two m, 1H each for 42D; chlorofusin = δ 3.42, t, 2H). This final multiplicity distinction between 40D and 42D allowed the absolute stereochemical assignment for the chlorofusin chromophore. This assignment of the absolute configuration for chlorofusin is especially significant given that both C4 configurations are found in azaphilone natural products with roughly equivalent representations.

An important 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 assigned to chlorofusin. Thus, regardless of the relative stereochemistry 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 characterization 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 this latter NOE did not prove diagnostic of a C4-Me/C8-H cis stereochemistry.

3.5 Total synthesis of chlorofusin and its 4R-Diastereomers

With the chromophore stereochemistry confidently assigned, the (4R,8S,9R)-diastereomer 40D was incorporated into a total synthesis of chlorofusin. Peptide 44, synthesized in a convergent sequence, was protected in a manner that avoids treatment of late stage chromophore derived intermediates with strong acid that might promote C9 N,O-spiroketal isomerization. Fmoc deprotection of 40D (piperidine) and coupling of the free amine 43 with heptapeptide 44 (EDCI, HOAt, 55%) provided 45 (Scheme 4). Concurrent benzyl ester deprotection and Cbz removal (H2, Pd/C) followed by macrocyclization of 46 (EDCI, HOAt, 60%) afforded synthetic chlorofusin (1), which displayed spectroscopic properties indistinguishable from that 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 in related studies.44

Scheme 4
Completion of the total synthesis of chlorofusin.

In addition to the natural (4R,8S,9R)-diastereomer constituting chlorofusin, the (4R,8R,9R)-diastereomer 3 proposed by Williams as well as the (4R,8S,9S)- and (4R,8R,9S)-diastereomers of chlorofusin (48 and 49) were also prepared by this route from 40A-40C. Notably, this entailed simply the three steps of Fmoc deprotection of the chromophore dipeptide precursor and EDCI-promoted coupling with the heptapeptide 44, concurrent Cbz and benzyl ester deprotection, and a final macrolactamization to provide diastereomers 3, 48 and 49 with purification of a single intermediate in addition to the final products (Scheme 5). This late stage divergence in a convergent total synthesis facilitated the assembly of the collective set of diastereomers and the anticipated non-correlation of their spectroscopic properties with that reported for the natural product provided further support for the new structural assignment.

Scheme 5
Synthesis of the three remaining 4R-diastereomers of chlorofusin. The table shows the average variation in total and diagnostic chemical shifts in proton and carbon spectra when compared with Williams’ original data.23

Following the completion of this work and the chlorofusin structural reassignment, the comparison of a sample of authentic chlorofusin (ca. 1 mg, aged and of unknown quality) provided by Dr. Stephen Wrigley in 2003 exhibited a CD spectrum indistinguishable (sign and magnitude) from synthetic 1 confirming the absolute configuration assignment (Fig. 9B). Moreover, its CD spectrum was readily distinguishable 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 49 (Figure 10A).

Fig. 9
(A) CD spectra (0.2 mM in MeOH) of 3, 48, 49 and 1, and (B) CD spectra of synthetic versus natural chlorofusin.
Fig. 10
1H NMR overlay of natural chlorofusin with all four C4-R chlorofusin chromophore diastereomers.

Just as significantly, the sample provided an 1H NMR spectrum of a sufficient quality to confirm that it represented the authentic natural product. Illustrated in Fig. 10 is the spectral overlay of a diagnostic region of the 1H NMR spectra of this sample of natural 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, including the syn diastereomer 3 proposed by Williams. Additionally, the comparisons in this region of the 1H NMR spectra confirmed 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.

3.6 Synthesis of all four 4S-diastereomers of chlorofusin

With the disclosure by Yao of the synthesis and miscorrelation of the (4S,8R,9S)-diastereomer 52 with chlorofusin,44 studies were extended to include the preparation and characterization of all four 4S-chromophore diastereomers of chlorofusin including 52. The preparation of this enantiomeric chromophore series simply required the late stage individual incorporation of 42A-42D into the full chlorofusin structure and again benefited from the convergent nature of the total synthesis providing diastereomers 2, 50, 51 and 52 in four steps of which 52 corresponds to the Yao structure (Scheme 6).

Scheme 6
Synthesis of the four 4S-diastereomers of chlorofusin.

With 2, 50, 51 and 52 in hand, their spectroscopic properties proved readily distinguishable from the natural product. Each displayed a long wavelength negative Cotton effect opposite that observed with chlorofusin. 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.

Similarly, the 1H NMR and 13C NMR spectroscopic properties of each diastereomer 2, 50, 51 and 52 were distinguishable from those of chlorofusin. This is most apparent in the region represented with the spectral overlay with the natural product in Fig. 11. The Yao diastereomer 52, bearing the enantiomer of the chromophore found in natural chlorofusin, is most readily distinguishable by its ornithine CH2δ 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 α-CH (δ 4.56 vs 4.59), Leu5 α-CH (δ 4.45 vs 4.48), Thr1 α-CH (δ 3.68 vs 3.66), Asn3 β-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 the natural product.

Fig. 11
1H NMR overlay of natural chlorofusin with all four C4-S chlorofusin chromophore diastereomers.

4 Conclusions

The first total synthesis of chlorofusin was achieved 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 diastereomers 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 properties indistinguishable from the natural product, but also all seven alternative chlorofusin chromophore diastereomers, each of which displayed spectroscopic properties readily distinguishable from the natural product. This included the diastereomer advanced by Yao44 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 characterization 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. One of the most interesting aspects of the contrathermodynamic N,O-ketal stereochemistry found in the chlorofusin chromophore is the prospect now that that other chlorofusin chromophore diastereomers, e.g. its more stable syn isomer 48, may likely be identified as natural products. The results of such studies as well as efforts to probe the mechanism by which chlorofusin disrupts the interaction of p53 and MDM2 will be facilitated by the work summarized herein.

5 Acknowledgments

We gratefully acknowledge the financial support of the National Institutes of Health (CA 41101), the Skaggs Institute for Chemical Biology and the Association for International Cancer Research (09-0166). RCC and SYL were Skaggs Fellows.


2-aminodecanoic acid
specific rotation at 23 °C and wavelength of sodium D line
circular dichroism
Correlation Spectroscopy (usually 1H, 1H)
camphorsulfonic acid
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
enantiomeric excess
Enzyme Linked Immunosorbent Assay
6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3, 5-octanedionate
Hep G2
human hepatocellular carcinoma cell line
O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
human double minute 2 protein
heteronuclear multiple bond correlation (usually 13C, 1H)
heteronuclear multiple quantum correlation (usually 13C, 1H)
o-iodoxybenzoic acid
methylaluminium bis(2,6-diphenylphenoxide)
murine double minute 2 protein
nuclear Overhauser effect
nuclear Overhauser effect spectroscopy
N-methylmorpholine oxide
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
rotating Overhauser effect spectroscopy
trifluoroacetic acid
p-toluenesulfonic acid

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