An approach to the site-selective diversification of apoptolidin A with peptide-based catalysts

Article abstract of DOI:10.1021/np9004932

We report the application of peptide-based catalysts to the site-selective modification of apoptolidin A (1), an agent that displays remarkable selectivity for inducing apoptosis in E1A-transformed cell lines. Key to the approach was the development of an

Full text of DOI:10.1021/np9004932

1864
J. Nat. Prod. 2009, 72, 1864–1869
An Approach to the Site-Selective Diversification of Apoptolidin A with Peptide-Based Catalysts
Chad A. Lewis,† Kate E. Longcore,‡ Scott J. Miller,*,† and Paul A. Wender*,‡
Department of Chemistry, Yale UniVersity, 225 Prospect Street, New HaVen, Connecticut 06520-8107, and Department of Chemistry and
Department of Chemical and Systems Biology, Stanford UniVersity, Stanford, California 94305-5080
ReceiVed August 9, 2009
We report the application of peptide-based catalysts to the site-selective modification of apoptolidin A (1), an agent that
displays remarkable selectivity for inducing apoptosis in E1A-transformed cell lines. Key to the approach was the
development of an assay suitable for the screening of dozens of catalysts in parallel reactions that could be conducted
using only microgram quantities of the starting material. Employing this assay, catalysts (e.g., 11 and ent-11) were
identified that afforded unique product distributions, distinct from the product mixtures produced when a simple catalyst
(N,N-dimethyl-4-aminopyridine (10)) was employed. Preparative reactions were then carried out with the preferred
catalysts so that unique, homogeneous apoptolidin analogues could be isolated and characterized. From these studies,
three new apoptolidin analogues were obtained (12-14), each differing from the other in either the location of acyl
group substituents or the number of acetate groups appended to the natural product scaffold. Biological evaluation of
the new apoptolidin analogues was then conducted using growth inhibition assays based on the H292 human lung
carcinoma cell line. The new analogues exhibited activities comparable to apoptolidin A.
Apoptolidin A (1) was isolated by Seto and co-workers from
the fermentation of the actinomycete Nocardiopsis sp. on the basis
of its selectivity for inducing apoptosis in E1A-transformed cells.1
Notably, apoptolidin is at least 4 orders of magnitude more active
in E1A-transformed cells than in nontransformed cells. Due to its
exquisite selectivity and the medicinal potential that such selectivity
holds, apoptolidin A (1) has been the focus of much synthetic
interest,2 and several derivatives involving modification of its
hydroxy groups, deglycosylation, and cycloadduct formation have
been reported.3 Evaluation of the activity of these derivatives has
provided a foundation for understanding how the structural features
of apoptolidin influence its functional activity including selective
induction of apoptosis in E1A-transformed 3Y1 rat fibroblasts,
inhibition of isolated F0F1-ATPase, and growth inhibition of human-
derived cancer cells. These studies have been further augmented
by the isolation of apoptolidins B, C, and D.4
Table 1. Assay Results with Apoptolidin Analogues (modified
from ref 3f)
GI50 (µM)
AD12-3Y1
GI50 (µM)
3Y1
IC50 (µM)
FoF1-ATPase
compound
apoptolidin A
C2′-OBz
1
2
3
4
5
6
7
8
9
0.0065
0.0036
0.0095
0.0098
0.056
0.0027
0.011
0.012
>1.0
>1.0
>0.6
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
0.7
0.3
0.4
0.8
0.8
0.4
1.1
2.8
17
C4′′′-OAc, C23-OAc
C4′′′-OAc
C16-OAc
C3′-OAc
C20-OAc
C20-OMe
isoapoptolidin
0.009
analogues 2-8 proved effective in obtaining new derivatives for
biological evaluation, the site of modification of apoptolidin in these
studies is controlled, and therefore limited, by the intrinsic reactivity
of the starting substrate. A complementary approach to analogues
would exploit catalyst-controlled, site-selective functionalization of
the complex natural product itself.5 While enzymes can often be
used for this purpose, application of readily available and varied
small-molecule catalysts offers another effective modification
strategy. Peptide-based catalysts, for example, have been used for
the site-selective modification of erythromycin A,6 producing novel
analogues in sufficient quantity so that biological studies could be
undertaken.7 The application of this approach to the apoptolidin
scaffold is reported herein, offering a one-step, organocatalytic route
to new biologically active apoptolidin derivatives.
Apoptolidin presents a special challenge to a catalyst-based
diversification strategy, as it possesses eight hydroxy groups, six
of which are secondary. Moreover, whereas a structure such as
erythromycin is readily available on a multigram scale, apoptolidin,
at the outset of this study, was available in only multimilligram
quantities. While a scaled-up fermentation could readily solve the
supply issue given recent improvements in the titer of apoptolidin
(ca. 150 mg/L),4a we elected to screen small-molecule catalysts
for selective derivatization of 1 with only ∼25 mg of apoptolidin
as the available starting material, as it represents a more commonly
encountered situation for which this approach would be used. In
order to assess feasibility, we chose to study catalytic acyl transfer
as the reaction of interest. If successful, such an approach could
help to validate the application of the small-molecule catalyst library
methodology to the derivatization of complex natural products, often
Previously, the Wender group produced a number of apoptolidin
A analogues through a progressive protection, functionalization,
and deprotection sequence.3d The products obtained from this three-
step sequence were tested for cell proliferation and mitochondrial
F0F1-ATPase inhibition (Table 1).3f These results revealed that the
structural alterations in compounds 2-8, as well as isoapoptolidin
(9), resulted in subtle and quite varied biological effects. While
the stepwise functionalization of apoptolidin used to produce
* To whom correspondence should be addressed. (S.J.M.) Tel: 203-432-
9885. Fax: 203-436-4900. E-mail: scott.miller@yale.edu. (P.A.W.) Tel: 650-
723-0208. Fax: 650-725-0259. E-mail: wenderp@stanford.edu.
† Yale University.
‡ Stanford University.
10.1021/np9004932 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/21/2009

Site-SelectiVe DiVersification of Apoptolidin A
Journal of Natural Products, 2009, Vol. 72, No. 10 1865
Figure 1. Low-conversion reactions of apoptolidin A (1). (a) Reversed-phase HPLC trace of the reaction mixture when 10 is employed as
the catalyst. (b) Reversed-phase HPLC trace of the reaction mixture when 11 is employed as the catalyst.
in scarce supply, as an important avenue for study at the intersection
of the fields of catalysis and natural products science.8
tion, 0.590 mM). When DMAP (10) was employed as the control
catalyst in order to define the inherent selectivity profile for
unprotected 1, it was found that the inherent reactivities of the C2′-
and C3′-hydroxy groups were roughly equivalent, providing nearly
equimolar amounts of the corresponding acetates (6:12 ∼ 1:1;
Figure 1a). After screening 156 catalysts, peptide 11 was found to
provide a notable enhancement of production of 12, with a product
ratio of ∼1:4 (6:12, Figure 1b). Importantly, this selective peptide-
catalyzed reaction was readily reproduced on a larger scale (0.02
to 7.1 mg apoptolidin), and apoptolidin A C2′-O-acetate (12) was
isolated. Critical to our goals, monoacetate 12 proved to be a
previously unknown compound, while monoacetate 6 had been
accessed previously.3d The structure of 12 was secured by NMR,
and chemical shift perturbations were consistent with those observed
for the known C2′ benzoate analogue (2).3d Furthermore, 12 was
isolated in the quantity and purity needed for biological testing (Vide
infra).
Results and Discussion
In order to allow for a sufficiently thorough study of the peptide-
catalyzed acylation of apoptolidin A (1), an assay was developed
that would consume as little of 1 as possible but deliver sufficient
amounts of products for HPLC analysis. Catalytic reactions were
performed using 0.02 mg of apoptolidin dissolved in 30 µL of
chloroform. This design, which was executed through the prepara-
tion of stock solutions, allowed for screening of dozens of
structurally diverse peptide catalysts (∼150-160) and required a
total of only 5 mg of substrate. After quenching the reactions, the
crude mixtures were analyzed by analytical reversed-phase HPLC.
Reactions were defined as successful “hits” when the corresponding
HPLC traces revealed a peak pattern that was significantly different
from those observed with control reactions. Control reactions were
defined as those that afforded the product distributions based on
the inherent reactivity of the substrate under conditions of catalytic
acylation mediated by N,N-dimethyl-4-aminopyridine (DMAP) and
in the absence of peptide catalyst. For the functionalization of 1,
acetic anhydride was chosen as the acyl donor due to its simplicity
and the availability of some “authentic” acetyl derivatives of
apoptolidin.3d Several pre-existing peptide-catalyst libraries9 were
then examined for catalytic modification of 1. Experiments were
performed at two different levels of conversion: low-conversion
reactions were targeted to monoacetates of 1 that would otherwise
require multistep syntheses; high-conversion reactions were pro-
jected to deliver bis-, tris-, or other poly(acetates) that would
likewise be difficult to access by designed synthetic routes based
on conventional protection strategies. Indeed, the objective was to
arrive at structures that would be specific to catalysts. Follow-up
and scale-up studies were therefore projected to be straightforward,
provided the screening reactions proved reproducible.
We then turned our attention to the discovery of catalysts for the
selective formation of poly(acetate) analogues of apoptolidin A.
Experimentally, this study was conducted by increasing the concentra-
tion of acetic anhydride and triethylamine 5-fold each. An increase in
the peptide-catalyst loading to 25 mol % was also found to be beneficial
for access to the bis- and tris(acetates). In comparison to the control
reaction catalyzed by DMAP (10, Figure 2a), we found that several
of the peptide catalysts offered substantial perturbation of the reactivity
of the hydroxy group array of apoptolidin A. Among these, the
enantiomer of catalyst 11 (ent-11) provided the product distribution
shown in Figure 2b. Once again, scale-up of the ent-11-catalyzed
reaction revealed good reproducibility, enabling isolation of the major
peak for characterization.
In this case, the major product peak was found to be a mixture
of two distinct tris(acetate) compounds, which were further
separated by silica gel chromatography. The two new apoptolidin
A analogues were then studied by spectroscopic techniques,
including tandem mass spectrometry fragmentation and NMR
spectroscopy. The characteristic mass fragmentation patterns, along
with the marked downfield chemical shift of protons alpha to the
For the low-conversion reactions, acetic anhydride (100 equiv)
and triethylamine were introduced into the reaction mixtures along
with 10 mol % of peptide-based catalysts (total reaction concentra-

1866 Journal of Natural Products, 2009, Vol. 72, No. 10
Lewis et al.
Figure 2. High-conversion reactions of apoptolidin A (1). (a) Reversed-phase HPLC trace of the reaction mixture when 10 is employed as
the catalyst. (b) Reversed-phase HPLC trace of the reaction mixture when ent-11 is employed as the catalyst.
newly introduced acetates relative to the parent compound,10
allowed assignment of new structures 13 and 14 (Table 2).
to retain biological activity when the hydroxy group array is
modestly modified (Table 1). These findings indicate that either
the modifications do not interfere with the apoptolidin’s interaction
with its molecular target, proposed in part to be mitochondrial F0F1-
ATP synthase,11,3f or the analogues are converted back to apop-
tolidin, perhaps through the action of esterases, in cell-based assays.
Peptide-based catalysts have been used to afford differentially
functionalized analogues of apoptolidin A. The screening of catalyst
libraries was possible with substrate samples involving microgram
quantities of the polyol natural product. The reproducibility of the
results was excellent when screening experiments were scaled up
in order to allow isolation and characterization of apoptolidin
analogues. From these studies, three new compounds were obtained,
and their structures were determined. These new natural product
analogues were then evaluated in growth inhibition assays using
the H292 human lung carcinoma cell line. All three analogues
showed nanomolar potencies, consistent with other acylated apo-
ptolidin analogues. Overall, these studies provide encouraging
results that further demonstrate that small-molecule, nonenzymatic
catalysts can be useful tools in the selective modification of complex
molecules, providing a potentially powerful entry into structural
and functional space of relevance to natural product scaffolds. The
fidelity of the experiments conducted on a minute screening scale
and subsequently upon scale-up for new analogue isolation bode
well for execution of related protocols in integrated studies involving
natural products isolation, derivatization, biological evaluation, and
optimization.7 The step economy12 achieved with this diversification
strategy through avoidance of protecting group manipulations argues
further for its use in the modification of complex molecules
available in limited quantities.
A particularly significant issue in the generation of new analogues
of 1 employing any approach is the vulnerability of 1 toward
rearrangement to the ring-expanded variants related to isoapoptolidin
(9).3b,c Notably, each of the new structures obtained in this study
of catalytic derivatization provides NMR spectra consistent with
the parent structure of 1, rather than that of the rearranged isomer
9. In particular, as shown in Figure 3 (also Table 2), the C19 proton
and C25 proton exhibit characteristic chemical shifts associated with
each structure.
The three newly isolated apoptolidin A analogues (12-14) were
tested for growth inhibitory activity in H292 human lung carcinoma
cells (Table 3). As in cells treated with apoptolidin A, reduced cell
numbers were observed at higher concentrations of analogues. The
acetylated analogues demonstrated marginally less potent activity
than apoptolidin A; however, these findings are consistent with
previous observations, namely, that apoptolidin A analogues tend
Experimental Section
General Experimental Procedures. Proton NMR spectra were
recorded on either a Varian 500 or 600 MHz spectrometer. Proton
chemical shifts are reported in ppm (δ) relative to residual solvent peak
(CD3OD, 3.31 ppm). Data are reported as follows: chemical shift
(multiplicity [singlet (s), doublet (d), triplet (t), quartet (q), and multiplet
(m)], coupling constants [Hz], integration). NMR data were collected
at 25 °C. High-resolution and tandem mass spectrometry were
performed at the Stanford University Mass Spectrometry Facility.
Analytical thin-layer chromatography (TLC) was performed using silica
gel 60 Å F-254 precoated plates (0.25 mm thickness). Visualization
was accomplished after staining with cerium ammonium molybdenate
(CAM) solution. Flash column chromatography was performed using
silica gel 60 Å (32-63 µm).
Preparative reversed-phase HPLC was run on a Varian Pro-Star
chromatograph equipped with a single wavelength UV detector (230
nm). Analytical reversed-phase HPLC was performed on a Hewlett-

Site-SelectiVe DiVersification of Apoptolidin A
Journal of Natural Products, 2009, Vol. 72, No. 10 1867

1868 Journal of Natural Products, 2009, Vol. 72, No. 10
Lewis et al.
mL of RPMI supplemented with 10% fetal bovine serum, penicillin,
and streptomycin in a 96-well plate. Cells were allowed to settle and
adhere for 24 h at 37 °C in a 5% CO2 atmosphere before apoptolidin
A derivative additions were performed in triplicate from 4 mM stock
solutions in DMSO to give a maximum final DMSO concentration of
0.25% (10 mM to 0.5 nM in 3-fold dilutions). After 48 h, viable cell
number was quantified using thiazolyl blue tetrazolium bromide.13
Briefly, 10 µL of a 5 mg thiazolyl blue tetrazolium bromide per mL
culture media was added to each well and incubated at 37 °C and 5%
CO2 for 2.5 h, at which time the cells were lysed with 100 µL of a
detergent solution (10% Triton X 100 in 0.1 N HCl in 2-propanol).
Following thorough mixing of the cells via pipet, the plates were read
using a VERSAmax tunable microplate reader (Molecular Devices) with
SOFTmax Pro version 3.1.1 software. Plates were read at 570 nm and
subtracted at 690 nm. EC50 values were calculated by fitting data with
GraphPad Prism 4 (nonlinear regression, sigmoidal dose response). The
reported values are the average of three independent experiments, and
the error represents the standard error of the mean.
General Catalyst Screening Procedure for the Acetylation of
Apoptolidin A. A chloroform solution of apoptolidin A (0.02 mg, 17.7
nmol/20 µL) was treated with a solution of the predissolved catalyst
(0.004 mg, 20 µL, 25 mol % based upon average MW of 1000). Acetic
anhydride (0.166 nL in 10 µL, 100 equiv) and triethylamine (0.244 nL
in 10 µL, 100 equiv) were then introduced. The reaction vessel was
then wrapped in foil, and the reaction mixture was allowed to stir for
approximately 48 h before it was quenched with MeOH (5 µL). The
reaction mixture was then concentrated and subsequently redissolved
in a solution of CH3CN/H2O (1:1, 20 µL). The sample was then
analyzed by reversed-phase HPLC (Waters Xterra RP 18 3.5 µm, 3.0
× 100 mm), eluting initially with 70% H2O/CH3CN, applying a gradient
to 35% H2O/CH3CN over 60 min (flow rate, 0.75 mL/min). The
retention time of the peptide catalyst used for the reaction was also
determined independently using the same method. For higher conversion
screening of apoptolidin analogues, acetic anhydride and triethylamine
stoichiometries were increased to 500 equiv and the peptide loading
was increased to 25 mol %.
Figure 3. Diagnostic apoptolidin A (1) and isoapoptolidin (9) NMR
data (CD3OD solvent).
Apoptolidin A C2′-O-acetate (12). To a solution of apoptolidin A
(7.1 mg, 0.0063 mmol) in CHCl3 (18 mL) were added Ac2O (60 µL,
0.63 mmol), triethylamine (88 µL, 0.63 mmol), and catalyst 11 (2.7
mg, 0.0030 mmol). The reaction was deemed complete after 80 h as
determined by analytical HPLC. Methanol (10 mL) was then added,
and the reaction was concentrated. Purification by preparative HPLC
provided unreacted apoptolidin A (1) (0.5 mg, 7%), the C2′-O-acetate
12 (1.9 mg, 26%), and the C3′-O-acetate 6.
Table 3. Growth Inhibition Assay of New Apoptolidin Analogues
in H292 Cells
compound
EC50 (nM)
1
21 ( 4
12
13
14
60 ( 11
48 ( 15
48 ( 10
Apoptolidin A C2′-O-acetate (12): 1H NMR (500 MHz, CD3OD),
see Table 2; HRMS m/z 1193.6442 (calcd for C60H98O22 + Na,
1193.6447); Rf ) 0.35 (9:1 CHCl3/MeOH v/v).
Packard 1100 Series chromatograph equipped with a diode array
detector (230 nm). All reactions were carried out under a nitrogen
atmosphere employing oven- or flame-dried glassware. All solvents
were either distilled or obtained from passing through activated alumina.
Peptide Synthesis and Screens. Peptides were synthesized on solid
support using commercially available Wang polystyrene resin preloaded
with FMOC-protected amino acid. Couplings were performed using 5
equiv of amino acid derivative, 5 equiv of HBTU, and 10 equiv of
Hu¨nig’s base in DMF for 3 h. Deprotections were performed using
20% piperidine in DMF for 20 min. To minimize diketopiperazine
formation, dipeptides were deprotected using 50% piperidine in DMF
for 5 min. Peptides were cleaved from solid support using a mixture
of MeOH/DMF/NEt3 (9:1:1) for 5 days. The peptides were characterized
by electrospray mass spectrometry and used in parallel reaction screens
without further purification. Peptides of interest were purified by
reversed-phase chromatography on a Biotage SP4 using C18 silica gel.
The peptides were purified by a gradient of 55% MeOH/H2O and
increasing to 80% MeOH/H2O over 35 column volumes.
Apoptolidin A C3′-O-acetate (6): Spectroscopic data for monoac-
etate 6 matched those previously reported. See ref 3d.
Apoptolidin A C2′-O-C20-O-C4′′′-O-trisacetate (13) and Apop-
tolidin A C2′-O-C3′-O-C20-O-trisacetate (14). Apoptolidin A (28.5
mg, 24.5 µmol) was dissolved in CHCl3 (35 mL), and the peptide
catalyst ent-11 was added (5.7 mg, 6.3 µmol). Triethylamine (1.74 mL,
12.5 mmol) was then added and the solution allowed to stir for 10
min. Lastly, acetic anhydride (1.18 mL, 12.5 mmol) was added, and
the reaction was then covered in foil and stirred at room temperature.
The reaction was monitored by sampling of aliquots that were quenched
in MeOH and concentrated. After 44.5 h, the reaction was deemed
complete and was quenched with MeOH (4 mL). The reaction was
concentrated and then dissolved in 1:1 CH3CN/H2O. The crude mixture
was purified by semipreparative reversed-phase HPLC (Waters XTerra
column RP-18 10 µm, 7.8 × 150 mm) and lyophilized. This material
was then subjected to silica gel purification to separate the two
trisacetates, 13 and 14, using 95:5 CHCl3/MeOH. The fractions
containing products were concentrated, and a second purification by
silica gel yielded the desired products 13 (3.2 mg, 10%) and 14 (0.7
mg, 2%).
Isolation of Apoptolidin A. Apoptolidin A was isolated as previ-
ously reported.1a 4a Briefly, following fermentation and centrifuga-
,
tion of the producing organism, Nocardiopsis sp., EtOAc/brine extrac-
tion provided crude material. Partial purification via silica gel column
chromatography (10% MeOH/CHCl3) was followed by final purification
by preparative reversed-phase HPLC (Alltech Alltima column (length
250 mm, i.d. 22 mm) packed with C18 (10 µm) stationary phase,
30-80% CH3CN/H2O linear gradient in 20 min). Fractions containing
apoptolidin A were lyophilized to dryness.
Apoptolidin A C2′-O-C20-O-C4′′′-O-trisacetate (13): 1H NMR
(600 MHz, CD3OD), see Table 2; HRMS m/z 1277.6655 (calcd for
C64H102O24 + Na, 1277.6659); Rf ) 0.44 (9:1 CHCl3/MeOH v/v).
Apoptolidin A C2′-O-C3′-O-C20-O-trisacetate (14): 1H NMR (600
MHz, CD3OD), see Table 2; HRMS m/z 1277.6670 (calcd for
C64H102O24 + Na, 1277.6659); Rf ) 0.36 (9:1 CHCl3/MeOH v/v).
Growth Inhibition Assay. Cell proliferation assays using H292 cells
were performed by plating cells at a density of 1000 cells/well in 100

Site-SelectiVe DiVersification of Apoptolidin A
Journal of Natural Products, 2009, Vol. 72, No. 10 1869
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Acknowledgment. We are grateful to Dr. A. Morgan for some
preliminary studies. S.J.M. is grateful to the NIH-NIGMS for support
of this work (GM-068649). Additional financial support for this research
was provided by NIH grant CA31845 (P.A.W.) and the Office of
Technology Licensing Stanford Graduate Fellowship (K.E.L.). Prof.
D. Mochly-Rosen (Stanford University) is thanked for kindly providing
laboratory space and equipment for growth inhibition assays.
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Supporting Information Available: Spectroscopic data for new
analogues including 1H NMR, 1H-1H COSY, and tandem mass
spectrometry data. This material is available free of charge via the
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