A multiply convergent platform for the synthesis of trioxacarcins

Article abstract of DOI:10.1073/pnas.1015257108

Many first-line cancer drugs are natural products or are derived from them by chemical modification. The trioxacarcins are an emerging class of molecules of microbial origin with potent antiproliferative effects, which may derive from their ability to covalently modify duplex DNA. All trioxacarcins appear to be derivatives of a nonglycosylated natural product known as DC-45-A2. To explore the potential of the trioxacarcins for the development of smallmolecule drugs and probes, we have designed a synthetic strategy toward the trioxacarcin scaffold that enables access to both the natural trioxacarcins and nonnatural structural variants. Here, we report a synthetic route to DC-45-A2 froma differentially protected precursor, which in turn is assembled in just six steps from three components of similar structural complexity. The brevity of the sequence arises from strict adherence to a plan in which strategic bond-pair constructions are staged at or near the end of the synthetic route.

Full text of DOI:10.1073/pnas.1015257108

A multiply convergent platform for
the synthesis of trioxacarcins
Jakub Švenda, Nicholas Hill, and Andrew G. Myers1
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved November 29, 2010 (received for review October 15, 2010)
Many first-line cancer drugs are natural products or are derived
from them by chemical modification. The trioxacarcins are an emer-
ging class of molecules of microbial origin with potent antiprolifera-
tive effects, which may derive from their ability to covalently
modify duplex DNA. All trioxacarcins appear to be derivatives of
a nonglycosylated natural product known as DC-45-A2. To explore
the potential of the trioxacarcins for the development of small-
molecule drugs and probes, we have designed a synthetic strategy
toward the trioxacarcin scaffold that enables access to both the
natural trioxacarcins and nonnatural structural variants. Here, we
report a synthetic route to DC-45-A2 from a differentially protected
precursor, which in turn is assembled in just six steps from three
components of similar structural complexity. The brevity of the
sequence arises from strict adherence to a plan in which strategic
bond-pair constructions are staged at or near the end of the
synthetic route.
of similar complexity at or near the final step of the sequence.
Although the importance of convergence in synthesis has long
been appreciated (12–21), highly condensed polycyclic targets
such as the trioxacarcins frequently do not lend themselves to
convergent simplification of the type specified, and synthesis plans
that might meet this objective can be stereochemically ambiguous
and (partly as a consequence) may offer modest probability of
success. As an important counterpoint, however, the end value of
a route that successfully achieves a high degree of convergence by
bond-pair constructions between components of similar complex-
ity is made evident by the rapidity with which large numbers of
structural analogs can be prepared and by the diversity of com-
pounds that can be synthesized through variation of the coupling
partners (21–23).
To address natural and nonnatural trioxacarcins broadly, we
targeted for synthesis compound 2 and as its precursor the cyclic
siloxane 3, differentially and orthogonally hydroxyl-protected
derivatives of DC-45-A2 (1). By the retrosynthetic derivation
outlined in Fig. 2, we envisioned that target 3 could be assem-
bled in one step by a 1,3-dipolar cycloaddition reaction between
the carbonyl ylide intermediate 4 and the aromatic aldehyde 5
(24, 25). Four diastereomeric cycloadducts can arise from the
pairwise combinations of the two diastereofaces of reactants 4
and 5 (Fig. S1); only one (an endo-cycloadduct) is stereochemi-
cally congruent with natural trioxacarcins. Although each of
the >20 prior examples of 1,3-dipolar cycloaddition reactions
between a five-membered cyclic carbonyl ylide and an aromatic
aldehyde was shown to proceed with exo selectivity (25, 26), we
anticipated that different stereochemical outcomes might be
achieved in the proposed cycloaddition reaction by variation of
catalyst (both catalyzed and noncatalyzed carbonyl ylide–alde-
hyde cycloadditions are known) and/or substrate (such as by mod-
ification of the protective groups of the aldehyde component).
We imagined that the carbonyl ylide intermediate 4 would derive
in one step from the epoxy diazo diketone precursor 6, and that
the aromatic aldehyde coupling component 5 could be assembled
in a second convergent bond-pair construction, by base-promoted
cyclization of the cyanophthalide 7 with the substituted cyclohex-
enone 8. The three precursors 6, 7, and 8 were viewed to be com-
ponents of similar synthetic complexity, as determined by analysis
of the number of steps required for their preparation.
chemical synthesis ∣ DNA alkylation ∣ anticancer
he trioxacarcins are highly concatenated, densely oxygenated
T
molecules encoded within the genes of various streptomycetes
and potently inhibit the growth of cultured human cancer cell
lines (1–4). Approximately 20 individual trioxacarcins have been
structurally characterized; all appear to be built upon or closely
related to the natural product DC-45-A2 (1) (5). The diversity of
the trioxacarcin class is well represented by the four compounds
that are depicted in Fig. 1 alongside the anthracycline antibi-
otic daunomycin (daunorubicin, long used in chemotherapy for
acute myelogenous leukemias) and nogalamycin, presented in
a manner that emphasizes their structural similarities. The triox-
acarcins, however, have unique glycosylation patterns, contain a
distinct linear aromatic core, and bear as their most distinguish-
ing feature a highly unusual condensed polycyclic trisketal con-
taining a spiro-epoxide. The latter is implicated to be key to
the observed antiproliferative effects of the trioxacarcins, for it
has been shown to serve as the point of covalent attachment of
trioxacarcin A (6–8), the most potent family member in clono-
genic assays (4) (with subnanomolar IC70 values), to G residues
of duplex DNA. An X-ray crystallographic analysis of a 2∶1 com-
plex of trioxacarcin A and an 8-mer duplex DNA oligonucleotide
(8) reveals that the structure has features related to the (2∶1)
nogalamycin-DNA complex (9), with the linear aromatic cores
of both small molecules bound intercalatively through the base
stack and sugar residues positioned in the major and minor
grooves, but unlike nogalamycin, trioxacarcin A covalently modi-
fies the DNA duplex, by reaction of the spiro-epoxide with N7 of
a flanking G residue as nucleophile (8). Other structurally distinct
natural product families appear to function by pathways that may
be mechanistically related (10, 11).
Results and Discussion
The densely functionalized epoxy diazo diketone 6, the precursor
to the carbonyl ylide intermediate 4, was synthesized in eight steps,
as shown in Fig. 3 Left, beginning with a highly diastereoselective
Author contributions: J.Š., N.H., and A.G.M. designed research; J.Š. and N.H. performed
research; and J.Š., N.H., and A.G.M. wrote the paper.
To realize their full potential as chemotherapeutic agents and
to enable a broader study of the trioxacarcin class of DNA-binding
molecules, we sought to develop a synthetic route that would allow
for the preparation of a wide array of new trioxacarcins in amounts
sufficient for biological evaluation. Toward this end and as a stra-
tegic objective, we determined to develop a route that approached
maximal convergence, with the further specification that it be
optimized toward bond-pair constructions between components
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The crystallography, atomic coordinates, and structural factors have been
deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Centre,
Cambridge CB2 1EZ, United Kingdom (accession no. CCDC 788988).
1To whom correspondence should be addressed. E-mail: myers@chemistry.harvard.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015257108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1015257108
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Fig. 1. Representative trioxacarcins and the anthracycline antibiotics daunomycin and nogalamycin.
auxiliary-controlled Baylis–Hillman reaction between the Oppol-
zer sultam-derived acrylimide 9 (1 eq) and anhydrous 2,2-di-
methoxyacetaldehyde (3.0 eq) using 1,4-diazabicyclo[2.2.2]octane
(DABCO, 0.3 eq) as catalyst in dichloromethane at 23 °C (16 h).
The product of the reaction had incorporated two molecules of
2,2-dimethoxyacetaldehyde, and had expelled the sultam auxiliary
by internal lactonization, as anticipated based on precedent (27).
Methanolysis of the resulting lactone (triethylamine in methanol,
30 min, 23 °C) afforded the corresponding methyl ester (see
SI Appendix), shown to be ≥98% enantiomerically pure by capil-
lary GC analysis. Hydroxyl protection with tert-butyldimethylsilyl
trifluoromethanesulfonate (1.2 eq) and N, N-diisopropylethyla-
mine (1.5 eq) then afforded the tert-butyldimethylsilyl ether 10
(48% over three steps). Nucleophilic epoxidation of 10 at 0 °C
(4.5 h) in the presence of tert-butylhydroperoxide (2.0 eq) and
potassium tert-butoxide (0.1 eq) provided selectively the anti-
epoxy ester 11 in 81% yield (anti∶syn ¼ 13∶1) (28). Saponification
of ester 11 (THF-MeOH-H2O, LiOH, 0 °C) and activation of the
resulting carboxylic acid by treatment with isobutylchloroformate
(1.05 eq) in the presence of Et3N (1.1 eq) at −20 °C with gradual
warming to −10 °C followed by addition of a solution of diazo-
methane in ether (∼0.25 M, 2.0 eq) and further warming to
23 °C provided, after chromatographic purification on triethyla-
mine-deactivated silica gel, the epoxy diazo ketone 12 as a yellow
oil (74% yield, two steps). Exposure of this product to triethyla-
mine-trihydrofluoride in acetonitrile led to efficient cleavage of
the tert-butyldimethylsilyl protective group, affording the corre-
sponding secondary alcohol in nearly quantitative yield. Oxidation
with Dess–Martin periodinane (1.1 eq) buffered with sodium
bicarbonate then afforded the corresponding epoxy diazo dike-
CH
O
3
CH
O
3
O
CH
O
3
O
CH
H
OPMB
H
O
3
O
CH
O
3
CH
3
O
CH
H
OPMB
H
O
3
CH
O
3
H
H
H
O
H
O
O
OH
H
O
OH
O
OTBS
O
H
O
OTBS
2
An Orthogonally Protected
Trioxacarcin Precursor
(
-bond rotation)
O
CH
O
3
H
O
O
CH
O
3
CH
O
3
O
CH
H
CH
O
OPMB
H
3
3
H
O
Deprotection
O
CH
O
O
3
CH
CH
H
O
OPMB
H
3
3
H
H
O
O
CH
3
O
O
OTBS
H
Si
CH
OH
3
O
O
OTBS
CH
CH
3
3
CH
3
CH
3
Cyclic Siloxane 3
Bond-Pair
Construction
O
O
CH
O
3
O
CH
O
N
2
3
H
O
O
CH O
3
H
CH
O
3
O
Carbonyl Ylide 4
H
Epoxy Diazo Diketone 6
Bond-Pair
CH
CH
O
OPMB
H
3
3
Construction
O
OPMB
CN
O
CH
3
H
O
3
CH
+
O
O
OTBS
CH
OTBS
Si
3
CH
3
O
MOMO
O
CH
CH
3
3
CH
3
CH
3
Cyanophthalide 7
Cyclohexenone 8
Aromatic Aldehyde 5
Fig. 2. Target identification and retrosynthetic analysis.
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Švenda et al.

tone 6 as a light yellow oil in 77% yield (two steps). The epoxy
diazo diketone 6 can be stored for several months without decom-
position at −25 °C with care to exclude light.
cyanophthalide 7 in 77% yield by brief exposure to cyanotri-
methylsilane followed by extended treatment with glacial acetic
acid (30, 31).
The cyanophthalide 7 was prepared in eight steps by a highly
practical route (Fig. 3 Center). 4-Methylsalicylic acid (13, 50 g)
was transformed in a sequence of five chemical transformations
using conventional synthetic techniques and a single, final chro-
matographic purification step to afford the 3,4-disubstituted
salicylamide 15 in 74% yield (57 g). Protection of the phenolic
hydroxyl group as a methoxymethyl ether and subsequent direc-
ted metalation–formylation (29) provided the aldehyde 16 in
94% yield. The latter intermediate was then transformed into the
Two different routes were developed for the synthesis of the
cyclohexenone fragment 8. The preferred route, shown in Fig. 3
Right (see SI Appendix for an alternate route), employed L-malic
acid as starting material and proceeded through a known
four-step sequence to the lactone 17. The latter product was
transformed into the aldehyde 18 in 70% yield (two steps)
by Weinreb amide formation (32) followed by oxidation with
aqueous sodium hypochlorite (1.0 eq) in the presence of 2,2,6,
6-tetramethylpiperidine-1-oxyl (TEMPO, 0.01 eq) and potassium
Fig. 3. A multiply convergent route for the synthesis of trioxacarcins.
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bromide (0.1 eq). Addition of aldehyde 18 to a 1∶1 mixture of
divinylzinc (2.0 eq) and the amino alkoxide ligand 19 (2.0 eq)
at −70 °C provided the 4S-alcohol 20 selectively (diastereomeric
ratio 12∶1), by which we infer that the substrate had reacted via
bidendate coordination to the organometallic reagent (33). The
product was susceptible to lactonization and so was protected
immediately as the 4-methoxybenzyl ether (21) using 4-methox-
ybenzyl trichloroacetimidate (2.0 eq) and ScðOTfÞ3 as catalyst
(0.03 eq, 57% yield over two steps, 12∶1 mixture of C4 diaster-
eomers, 17.8-g scale). Addition of vinylmagnesium bromide
(3.0 eq) to amide 21 provided the divinyl ketone, which under-
went smooth ring-closing metathesis in the presence of the sec-
ond-generation Hoveyda–Grubbs ruthenium alkylidene catalyst
(34) at 55 °C, affording the diastereomerically pure cyclohexe-
none coupling component 8 in 75% yield after chromatographic
purification.
In the first of two late-stage ring-forming (bond-pair) coupling
reactions, components 7 and 8 were combined in a Kraus–Sugi-
moto cyanophthalide annulation reaction (35). Thus, addition of
a solution of enone 8 (1 eq) to a cold solution of the cyanophtha-
lide anion obtained by deprotonation of 7 (1.0 eq) with lithium
tert-butoxide (3.0 eq) in tetrahydrofuran at −78 °C led to rapid
formation of Michael addition product(s) (based on TLC analysis,
not characterized); upon warming to −40 °C these underwent
cyclization to form a single dihydroquinone phenolate, which was
trapped in situ by monomethylation with dimethylsulfate (−26 →
23 °C). The anthrone methyl ether 22 was obtained in 57% yield
(3-g scale, yellow foam) after chromatographic isolation, a process
facilitated by the long-wavelength UVabsorption of the product,
with blue-green fluorescence, a characteristic of the trioxacarcins.
Oxidative cleavage of the alkenyl side chain at 0 °C (NaIO4,
K2OsO4·2H2O, 2,6-lutidine) (36) then provided the correspond-
ing aldehyde as an orange foam (69% yield). The methoxymethyl
(MOM) protective group was selectively removed upon treat-
ment of the latter product with B-bromocatecholborane (2.0 eq),
affording an air-sensitive bisphenol intermediate (89% yield) as
a yellow foam. Silylation (t-Bu2SiCl2, HOBt, DIPEA, 55 °C)
(37) then provided the much more stable di-tert-butylsiloxane
derivative 5 (50% yield), the substrate for final coupling.
The fully oxygenated polycyclic skeleton of the trioxacarcins
was assembled in one step by slow addition (syringe pump, 2 h)
of a solution of the epoxy diazo diketone 6 (3.0 eq, 2.26 M in
dichloromethane) to a stirring suspension of aldehyde 5 (1 eq,
0.75 M in dichloromethane), rhodium(II) acetate (0.05 eq), and
powdered, activated 4 Å molecular sieves at 23 °C. After filtration
to remove the rhodium catalyst, the filtrate was concentrated,
and the residue was purified by RP-HPLC to provide in 63%
yield a mixture of diastereomeric cycloadducts in which the two
endo-diastereomers (3 and 23) greatly predominated (Fig. 3 and
S1 Appendix). Pure samples of the individual diastereomers were
obtained for spectroscopic analysis (see SI Appendix), but for
preparative purposes it proved to be much more practical to
separate the diastereomers after cleavage of the cyclic di-tert-
butylsiloxane protective group (triethylamine-trihydrofluoride,
23 °C, 15 min), where the endo-diastereomers alone underwent
spontaneous hemiketalization; these products, both obtained as
bright yellow oils, were easily separated by RP-HPLC [24, 52 mg
(36% yield) and 2, 48 mg (34% yield)]. Variation of the catalyst
was indeed found to greatly influence the stereochemical out-
come of the cycloaddition. For example, cycloaddition of 5 and
6 in the presence of copper(I) tetrakis(acetonitrile) afforded as
the major product an exo-diastereomer (46%) that represented
only 14% of the diastereomeric product distribution when rho-
dium(II) acetate was used as catalyst. Although we believe it
likely that the efficiency and stereoselectivity of formation of
the desired endo-cycloadduct (3) may be improved by further
exploration of different catalysts, in its present form the rho-
dium(II) acetate-catalyzed transformation provides more than
sufficient quantities of material for biological evaluation and
mechanistic study. Two-step deprotection of endo-hemiketal
2
(DDQ; triethylamine-trihydrofluoride) afforded synthetic
DC-45-A2 (1) as a bright yellow powder (23 mg, 70% yield).
Spectroscopic data for the synthetic substance were fully consis-
tent with those reported for the natural product (5). Crystalliza-
tion of synthetic DC-45-A2 from ethyl acetate-hexanes provided
a single crystal suitable for X-ray diffraction analysis; two repre-
sentations of the three-dimensional structure obtained are
depicted in Fig. 3. This structure conforms fully with that pro-
posed for the natural product (5) and shows that the spiro-epox-
ide is ideally aligned for opening by a G residue stacked upon the
π-face of the tricyclic core. It is revealing that similar two-step
deprotection of the stereoisomeric endo-hemiketal 24 (with the
more electrophilic carbon of the spiro-epoxide oriented away
from the tricyclic aromatic core) gave rise to a chlorohydrin
derivative (25%), presumably arising from ring-opening of the
spiro-epoxide by chloride ion during workup, as well as the
expected spiro-epoxide, iso-DC-45-A2 (25, 22%, depicted in
Fig. 4 A). No such opening was observed with DC-45-A2. In con-
trast, in experiments evaluating the reactions of DC-45-A2 (1)
and iso-DC-45-A2 with the G residue of a known DNA substrate
for alkylation by trioxacarcin A (7), iso-DC-45-A2 was found to
be unreactive, whereas 1 readily alkylated the DNA duplex, as
discussed below.
We measured IC50 values of DC-45-A2, iso-DC-45-A2, and a
fully synthetic analog, dideoxy-DC-45-A2 (26), which we pre-
pared by the six-step route outlined in Fig. 3 without variation,
save for the use of 2-cyclohexen-1-one as starting material in
place of the substituted cyclohexenone coupling component 8,
in HeLa and H460 cell lines (Fig. 4 A). DC-45-A2 inhibited the
growth of both cell lines at micromolar concentrations. Dideoxy-
DC-45-A2 was found to be a more potent growth inhibitor, with
submicromolar IC50 values, and iso-DC-45-A2 was found to
be inactive. Both DC-45-A2 and dideoxy-DC-45-A2 were found
to modify a self-complementary 12-mer duplex oligonucleotide
containing a single (central) G residue (7) at 23 °C, as determined
by nondenaturing polyacrylamide gel electrophoresis with in-gel
fluorescence detection as well as liquid chromatography–mass
spectrometry (LC-MS) experiments, albeit with different rates
and efficiencies of alkylation (Fig. 4 B–D). Although alkylation
of the DNA duplex by DC-45-A2 proceeds with a half-life of
hours at 23 °C, the dideoxy-analog reacts with the DNA duplex
within minutes at 23 °C and with apparently greater efficiency
(Fig. 4 C and D). Iso-DC-45-A2 was not observed to modify
the same DNA duplex under any conditions examined.
Heretofore, antiproliferative effects of nonglycosylated triox-
acarcins such as DC-45-A2 have not been reported, so far as
we are aware, nor has their chemistry with deoxyribonucleic acids
been studied. Our findings suggest that the nonglycosylated, rigid
polycyclic framework of DC-45-A2, with a naturally configured
spiro-epoxide function, comprises structural features necessary
and sufficient to provide an electrophile capable of alkylating
G residues of duplex DNA, and that substantial variation in the
rate, efficiency, and perhaps sequence specificity of DNA alkyla-
tion (not evaluated here) might be achieved by substitution upon
this framework, which need not necessarily involve glycosylation.
Structural variations by the convergent route reported can be
achieved in two distinct ways, which together should allow for
multiplicative enhancement of the pool of synthetic trioxacarcins.
First, selective derivatization of the hydroxyl groups should be
feasible by virtue of their orthogonal protection. Second, more
deep-seated structural changes can be achieved by variation of
any of the three coupling components [exemplified by the synth-
esis of dideoxy-DC-45-A2 (26) above]. We believe that the route
to trioxacarcins described enables a comprehensive and broad
evaluation of trioxacarcin-based structures as potential che-
motherapeutic agents and provides a viable basis for their pro-
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Fig. 4. Synthetic nonglycosylated trioxacarcins, their antiproliferative activities in cultured human cancer cells, and DNA-modifying effects. (A) IC50 values for
DC-45-A2, iso-DC-45-A2, and dideoxy-DC-45-A2 measured in HeLa (cervical cancer) and H460 (lung cancer) cells lines. (B) Images of TBE 20% polyacrylamide gels
of the products of the reaction of the self-complementary duplex 12-mer d(AATTACGTAATT) (100 μM) with DC-45-A2 (lane 2, 100 μM), iso-DC-45-A2 (lane 3,
100 μM), or dideoxy-DC-45-A2 (lane 4, 100 μM) for 2 h at 23 °C; visualized with ethidium bromide and by in-gel fluorescence. (C) Images of TBE gels of the
products of the reaction of the self-complementary duplex 12-mer d(AATTACGTAATT) (100 μM) with DC-45-A2 or dideoxy-DC-45-A2 (25 μM) at 23 °C for the
indicated times; visualized by in-gel fluorescence. (D) LC-MS chromatograms of reaction mixtures of the self-complementary duplex 12-mer d(AATTACGTAATT)
(100 μM) and (i) DC-45-A2 (100 μM), (ii) iso-DC-45-A2 (100 μM), or (iii) dideoxy-DC-45-A2 (100 μM) after 24 h at 23 °C; iv depicts the LC-MS chromatogram of
the reaction mixture of the self-complementary duplex 12-mer d(AATTACGTAATT) (100 μM) and dideoxy-DC-45-A2 (100 μM) after 24 h at 23 °C followed by
the addition of piperidine (1 M) and heating for 30 min at 95 °C; ⦁, (AATTACGTAATT); □, (AATTACGTAATT • 1); ♦, (AATTACGTAATT • 26); ▪, (pTAATT);
○= (AATTACp).
was filtered, and the filtrate was concentrated. The residue (1 eq, see above)
was dissolved in acetonitrile (1.3 mL), and triethylamine-trihydrofluoride
(208 μL, 1.28 mmol, 20 eq) was added at 23 °C. The reaction flask was covered
duction on scales necessary to support clinical evaluation should
preclinical studies support such advancement.
with aluminum foil to exclude light. After 13.5 h, the reaction mixture was
Materials and Methods
partitioned between saturated aqueous sodium chloride solution (10 mL)
and dichloromethane (50 mL). The layers were separated. The organic layer
was dried over sodium sulfate. The dried solution was filtered, and the
filtrate was concentrated. The residue was purified by preparatory HPLC
to provide 23 mg of the product, DC-45-A2 (1), as a bright yellow pow-
der (70%).
General. Experimental procedures and spectral data for all compounds
created for this study can be found in the SI Appendix. The experimental
details, geometric parameters, perspective views, and three-dimensional
supramolecular architecture associated with the X-ray crystal structure of
DC-45-A2 are provided in Tables S1–S3 and Figs. S2 and S3 of the SI Appendix.
Preparation of DC-45-A2 (1). 2,3-Dichloro-5,6-dicyanobenzoquinone (17.4 mg,
76.7 μmol, 1.2 eq) was added to a vigorously stirring, biphasic solution of
differentially protected DC-45-A2 (2) (48 mg, 63.8 μmol, 1 eq) in dichloro-
methane (1.3 mL) and water (130 μL) at 23 °C. The reaction flask was covered
with aluminum foil to exclude light. Over the course of 5 h, the reaction
mixture was observed to change from myrtle green to lemon yellow. The
product solution was partitioned between saturated aqueous sodium
chloride solution (5 mL) and dichloromethane (50 mL). The layers were sepa-
rated. The organic layer was dried over sodium sulfate. The dried solution
ACKNOWLEDGMENTS. J.S. acknowledges Dr. Alfred Bader for his creation
and generous financial support of the Alfred Bader Fellowship Program in
Chemistry at Harvard University. Dr. Robert Yu measured the IC50 values
of DC-45-A2, iso-DC-45-A2, and dideoxy-DC-45-A2, which we acknowledge
with appreciation. We thank Dr. Shao-Liang Zheng for his help with X-ray
data collection and structure determination. Financial support from the
National Institutes of Health (Grant CA047148) and the National Science
Foundation (Grant CHE-0749566) is gratefully acknowledged.
1. Tomita F, Tamaoki T, Morimoto M, Fujimoto K (1981) Trioxacarcins, novel antitumor
antibiotics I. Producing organism, fermentation and biological activities. J Antibiot
34:1519–1524.
2. Tamaoki T, Shirahata K, Iida T, Tomita F (1981) Trioxacarcins, novel antitumor
antibiotics II. Isolation, physico-chemical properties and mode of action. J Antibiot
34:1525–1530.
Švenda et al.
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3. Maiese WM, et al. (1990) LL-D49194 antibiotics, a novel family of antitumor agents:
Taxonomy, fermentation and biological properties. J Antibiot 43:253–257.
4. Maskey RP, et al. (2004) Anti-cancer and antibacterial trioxacarcins with high anti-
malaria activity from a marine streptomycete and their absolute stereochemistry.
J Antibiot 57:771–779.
21. Nielsen TE, Schreiber SL (2008) Towards the optimal screening collection: A synthesis
strategy. Angew Chem Int Edit 47:48–56.
22. Charest MG, Lerner CD, Brubaker JD, Siegel DR, Myers AG (2005) A convergent
enantioselective route to structurally diverse 6-deoxytetracycline antibiotics.
Science 308:395–398.
23. Sun C, et al. (2008) A robust platform for the synthesis of new tetracycline antibiotics.
J Am Chem Soc 130:17913–17927.
24. Huisgen R (1963) 1,3-Dipolar cycloadditions past and future. Angew Chem Int Edit
2:565–598.
25. Padwa A, Chinn RL, Hornbuckle SF, Zhi L (1989) Cyclic carbonyl ylide formation from
the rhodium(II) acetate catalyzed reaction of 1-diazoalkanediones. Tetrahedron Lett
30:301–304.
26. Muthusamy S, et al. (2002) Tandem cyclization-cycloaddition behavior of rhodium
carbenoids with carbonyl compounds: Stereoselective studies on the construction
of novel epoxy-bridged tetrahydropyranone frameworks. J Org Chem 67:8019–8033.
27. Brzezinski LJ, Rafel S, Leahy JW (1997) The asymmetric Baylis–Hillman reaction. J Am
Chem Soc 119:4317–4318.
28. Švenda J, Myers AG (2009) Anti-selective epoxidation of methyl α-methylene-β-tert-
butyldimethylsilyloxycarboxylate esters. Evidence for stereospecific oxygen atom
transfer in a nucleophilic epoxidation process. Org Lett 11:2437–2440.
29. de Silva SO, Reed JN, Snieckus V (1978) Directed lithiation of N,N-diethylbenzamides.
Regiospecific synthesis of contiguously tri- and tetra-substituted alkoxybenzenes.
Tetrahedron Lett 19:5099–5102.
30. Freskos JN, Morrow GW, Swenton JS (1985) Synthesis of functionalized hydroxyphtha-
lides and their conversion to 3-cyano-1(3H)-isobenzofuranones. The Diels–Alder reac-
tion of methyl 4,4-diethoxybutynoate and cyclohexadienes. J Org Chem 50:805–810.
5. Shirahata K, Iida
T (1984) Compounds having antibiotic activity, processes for
their preparation, pharmaceutical compositions containing them and their use as
medicaments. US Patent 4,459,291.
6. Maskey RP, Sevvana M, Usón I, Helmke E, Laatsch H (2004) Gutingimycin: A highly com-
plex metabolite from a marine streptomycete. Angew Chem Int Edit 43:1281–1283.
7. Fitzner A, Frauendorf H, Laatsch H, Diederichsen U (2008) Formation of gutingimycin:
Analytical investigation of trioxacarcin A-mediated alkylation of dsDNA. Anal Bioanal
Chem 390:1139–1147.
8. Pfoh R, Laatsch H, Sheldrick GM (2008) Crystal structure of trioxacarcin A covalently
bound to DNA. Nucleic Acids Res 36:3508–3514.
9. Smith CK, Davies GJ, Dodson EJ, Moore MH (1995) DNA-nogalamycin interactions: The
crystal structure of d(TGATCA) complexed with nogalamycin. Biochemistry
34:415–425.
10. Sun D, Hansen M, Clement JJ, Hurley LH (1993) Structure of the altromycin B (N7-gua-
nine)-DNA adduct. A proposed prototypic DNA adduct structure for the pluramycin
antitumor antibiotics. Biochemistry 32:8068–8074.
11. Tse WC, Boger DL (2004) Sequence-selective DNA recognition: Natural products and
nature’s lessons. Chem Biol 11:1607–1617.
12. Velluz L, Valls H, Nominé G (1965) Recent advances in the total synthesis of steroids.
Angew Chem Int Edit 4:181–200.
13. Hendrickson JB (1977) Systematic synthesis design. 6. Yield analysis and convergency.
J Am Chem Soc 99:5439–5450.
14. Evans DA, Bender SL, Morris J (1988) Total synthesis of the polyether antibiotic X-206.
J Am Chem Soc 110:2506–2526.
15. Kaiser ET, et al. (1989) Peptide and protein synthesis by segment synthesis-condensa-
tion. Science 243:187–192.
16. Armstrong RW, et al. (1989) Total synthesis of a fully protected palytoxin carboxylic
acid. J Am Chem Soc 111:7525–7530.
17. Hitchcock SA, Boyer SH, Chu-Moyer MY, Olson SH, Danishefsky SJ (1994) A convergent
total synthesis of calicheamicin γI1. Angew Chem Int Edit 33:858–862.
18. Nicolaou KC, et al. (1998) Total synthesis of brevetoxin A. Nature 392:264–269.
19. Smith AB, III, Kaufman MD, Beauchamp TJ, LaMarche MJ, Arimoto H (1999) Gram-scale
synthesis of (+)-discodermolide. Org Lett 1:1823–1826.
20. Warren JD, Miller JS, Keding SJ, Danishefsky SJ (2004) Towards fully synthetic
glycoproteins by ultimately convergent routes: A solution to a long-standing problem.
J Am Chem Soc 126:6576–6578.
31. Nomura K, Okazaki K, Hori K, Yoshii
E (1986) Cyanophthalide annulation with
4-(5-alkoxy-2-furyl)-3-buten-2-one. Application to the synthesis of a (naphtho)pyra-
no-γ-lactone. Chem Pharm Bull 34:3175–3182.
32. Williams JM, et al. (1995) A new general method for preparation of N-methoxy-N-
methylamides. Application in direct conversion of an ester to a ketone. Tetrahedron
Lett 36:5461–5464.
33. Brubaker JD, Myers AG (2007) A practical, enantioselective synthetic route to a key
precursor to the tetracycline antibiotics. Org Lett 9:3523–3525.
34. Kingsbury JS, Harrity JPA, Bonitatebus PJ, Hoveyda AH (1999) A recyclable Ru-based
metathesis catalyst. J Am Chem Soc 121:791–799.
35. Kraus GA, Sugimoto H (1978) An annelation route to quinones. Tetrahedron Lett
19:2263–2266.
36. Yu W, Mei Y, Kang Y, Hua Z, Jin Z (2004) Improved procedure for the oxidative cleavage
of olefins by OsO4-NaIO4. Org Lett 6:3217–3219.
37. Trost BM, Caldwell CG (1981) The di-t-butylsilylene protecting group for diols.
Tetrahedron Lett 22:4999–5002.
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