Convenient synthesis of NCS - Chromophore metabolite isosteres: Binding agents for bulged DNA microenvironments

Article abstract of DOI:10.1021/ol0400591

(Chemical Equation Presented) A designed molecule with capacity to bind DNA bulges (20) has been prepared from readily available starting materials. The spirocyclic template was modeled on a metabolite of neocarzinostatin chromophore (NCSi-gb) and is equi

Full text of DOI:10.1021/ol0400591

ORGANIC
LETTERS
2005
Vol. 7, No. 1
71-74
Convenient Synthesis of
NCS Chromophore Metabolite
−
Isosteres: Binding Agents for Bulged
DNA Microenvironments
Yiqing Lin† and Graham B. Jones*
Bioorganic and Medicinal Chemistry Laboratories, Department of Chemistry and
Chemical Biology, Northeastern UniVersity, Boston, Massachusetts 02115
Geum-Sook Hwang,† Lizzy Kappen, and Irving H. Goldberg
Department of Biological Chemistry and Molecular Pharmacology,
HarVard Medical School, Boston, Massachussetts 02215
gr.jones@neu.edu
Received October 12, 2004
ABSTRACT
A designed molecule with capacity to bind DNA bulges (20) has been prepared from readily available starting materials. The spirocyclic
template was modeled on a metabolite of neocarzinostatin chromophore (NCSi-gb) and is equipped with functionality to enable convenient
bioassay. Preliminary studies confirm binding at specific bulged sequences and induction of polymerase-mediated slippage events. The target
compound offers a convenient means to study affinity for unique bulged motifs and for use as a molecular biology reagent.
Enediyne antibiotics have been the focus of intense research
over the past decade due to their observed antitumoral
activity and unique modes of action.1 Clinical candidates
have now emerged fueling additional interest in the molecular
mechanisms of this intriguing class, whose activity is derived
in part from an ability to induce strand breaks in duplex
DNA.2 Recent studies on the neocarzinostatin chromophore
[NCS-chrom], however, have revealed an alternate target and
mode of action.3 Specifically, the NCS-chrom phenoxide ion
has been shown to undergo a general base-catalyzed spiro-
lactonization and intramolecular rearangement to eneyne
allene 1, whose biological target is bulged DNA as opposed
to the duplex form.3 Following cycloaromatization of 1,
which proceeds via diradical intermediates capable of cleav-
ing bulged DNA, metabolite NCSi-gb is formed. Molecular
studies have revealed that the affinity of NCSi-gb to bind
DNA bulges is dependent on numerous features, including
the spirocyclic ring junction, pendant aminosugar group, and
two discrete aromatic moieties for π-stacking.3 Since bulged
* Fax: 617-373-8795.
† These authors contributed equally to this work.
(1) Jones, G. B.; Fouad, F. S. Curr. Pharm. Des. 2002, 8, 2415-2440.
(2) Xi, Z.; Goldberg, I. H. In ComprehensiVe Natural Product Chemistry;
Barton, D. H. R., Nakanishi, K., Meth-Cohn, O., Eds.; Elsevier: Oxford,
1999; Vol. 1, pp 553-592, .
(3) Stassinopoulos, A.; Ji, J.; Gao, S.; Goldberg, I. H. Science 1996, 272,
1943-1946. Kappen, L. S.; Goldberg, I. H. Science 1993, 261, 1319-
1321. Xi, Z.; Mao, Q. K.; Goldberg, I. H. Biochemistry 1999, 38, 4342-
4354.
10.1021/ol0400591 CCC: $30.25
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structures in nucleic acids are of general biological signifi-
cance (proposed as intermediates in RNA splicing, frame-
shift mutagenesis, intercalator-induced mutagenesis, and
imperfect homologous recombination and as binding motifs
for regulatory proteins),4 designed small molecules with
bulge affinity have become sought after targets.
Figure 1.
namically stable platform, as the spiro alcohol was prone to
retro-aldol reaction on decomposition. With these factors in
mind, a synthesis was devised to a functional core. 7-Meth-
Following qualitative SAR analysis of NCSi-gb, we designed
and synthesized spiro alcohol 2, the first lead compound for
library design.5 Crystallographic analysis of the core sup-
ported the design philosophy, the molecule adopting the
wedge-shape necessary to achieve binding to bulges (Figure
1). Simple aminosugar derivatives of the spirohydroxy group
showed good affinity for bulged sequences, but a number
of shortcomings led us to refine our design criterion viz. 2.5
Specifically, we desired (1) additional functionality to permit
(fluorescence) binding studies, (2) installation of the amino-
sugar in the appropriate location, and (3) a more thermody-
oxy tetralone was acetylated (3) and then converted to diene
4 (Scheme 1).6 Intermolecular cycloaddition with dienophile
5 (produced from tetrabromo-o-xylene) gave adduct 6 in
good yield. Reduction of the keto group at the locus of the
desired aminosugar (7) and subsequent inversion and mask-
ing (8) allowed us to use previously reported oxidative
cleavage followed by aldol chemistry to form spirocycles 9
for subsequent manipulation.5
Scheme 1. Preparation of Multiply Substituted Spirocyclic Building Blocks
72
Org. Lett., Vol. 7, No. 1, 2005

Scheme 2. Attempted Routes to Aminoglycosylated Enone
Scheme 3. Alkene Variant to Aminoglucosyl Enone
Core
Thwarted by this obstacle, an alternate masking group was
introduced. Specifically, 9a was methylated to 14, which
allowed unmasking of the benzoate ester to give 15; however,
all attempts to brominate R to the ketone resulted in
decomposition. Suspecting that the free benzyl alcohol was
playing a role, we investigated enone generation on the
masked substrate. However, in all cases the only product
isolated was inVerted stereoisomer 16, suggesting retro-aldol
reaction followed by reclosure onto a methylketonium ion.8
We initially elected to mask the spirohydroxy group as
the corresponding acetate, which allowed liberation of the
benzylic alcohol (10) (Scheme 2). Installation of the desired
enone group proceeded smoothly, as did aminoglycosylation
with 117 to produce advanced substrate 12. Though the Fmoc
group was removed without incident, all attempts to isolate
the product from global deacetylation failed, resulting in
isolation of 13 and associated decomposition products.
(5) Xi, Z.; Hwang, G.-S.; Goldberg, I. H.; Harris, J. L.; Pennington, W.
T.; Fouad, F. S.; Qabaja, G.; Wright, J. M.; Jones, G. B. Chem. Biol. 2002,
9, 925-931.
(6) Satisfactory spectroscopic and analytical data were obtained for all
new compounds.
(7) Produced from R-aminoglucose: FmocCl, NaHCO3, dioxane, water,
1 h, and then Ac2O, Py, 12 h (91%); N2H4, HOAc, DMF, 50 min, and then
Cl3CCN, DBU, CH2Cl2, 45 min (28%). Compound 12 was isolated as a
single diastereomer following separation after glycosylation. For coupling
methodology, see: Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl.
1980, 19, 731-732.
(4) Singer, R. H. Science 1998, 280, 696-697. Perutz, M. F. Curr. Opin.
Struct. Biol. 1996, 6, 848-858.
Org. Lett., Vol. 7, No. 1, 2005
73

routes to this class of agent. Indeed, for some substrates the
affinity of 20 is superior to the natural product metabolite
(entries 5 and 7), suggesting adoption of unique architectures.
Given the ease of access to 20, we became interested to gauge
effectiveness as a molecular biology tool. Bulged structures
have been proposed as intermediates in DNA slippage
synthesis involving the extension of nucleotide repeats. Given
the possible involvement of the slippage process in the
progression of certain neurological disorders (e.g., CAG
repeats in Huntington’s disease10), a readily available reagent
capable of inducing slippage may have a number of uses.
Accordingly, 20 was subjected to a slippage assay, which
measures 32P TMP uptake for DNA slippage synthesis
dependent on primers/templates bearing simple sequence
repeats, catalyzed by the Klenow fragment of DNA poly-
merase I. The results (Table 2) confirm that 20 can
significantly promote slippage within the 5 h assay with >10-
fold induction at 30 µM.11
Table 1. Dissociation Constant (µM) of Spirocyclic Alkenes
20 for Selected Sequencesa
Table 2. Effect of 20 on DNA Slippage Synthesisa
32P incorporated (CPM)
stimulation
control
20 (10 µM)
20 (30 µM)
38 664
89 101
511 667
2.3
13.2
a DNA slippage synthesis was measured using the primer/template
oligomers of thymidylate (20-mer) and deoxyadenylate (30-mer) in a
reaction catalyzed by the Klenow fragment of DNA polymerase I.10 The
annealed mixture of T20/A30 (2 µM) in 50 mM Tris-HCl pH 7.5 was
supplied with 5 mM magnesium, 4 mM dithiothreitol, 1 mM dATP, 0.5
mM TTP, and 1.5 µCi [R- 32P]TTP before starting the synthesis by the
addition of enzyme. Test compound was present from the beginning of the
reaction. After incubation at 37° for 5 h. 32P-TMP incorporated into acid-
precipitable DNA products was determined, and data were reported relative
to control.11
a Fluorescence quenching studies conducted using a SPEX Fluoromax-2
at 4 °C in phosphate buffer (10 mmol, pH 7.0). Emission spectra of 20 and
21 were obtained in the range 400-600 nm upon excitation at 385 nm.
Emission reading at 490 nm was imported in binding calculation. Dissocia-
tion constant (kd) was derived from curve-fitting (Kaleidagraph).5
Prompted by the retro-aldol issue, we made a decision to
investigate the effects of eliminating the spiro alcohol group
altogether. Though the crystal structure of 2 suggests that
hydrogen bonding of this group contributes to secondary
structure, the wedge-shaped template should persist on the
basis of the spiro junction. Accordingly, elimination of the
alcohol from 9a to give alkene 17 was effected (Scheme 3).
Conversion to enone 18 was uneventful as expected, and
saponification followed by aminoglycosylation gave 19 and
its stereoisomer in good yield. At this point, diastereomer
resolution of stereoisomers at the spiro junction was possible,
the desired isomer subjected to deprotection to give NCSi-
gb mimic 20. To provide a convenient control substrate, the
spiro-inverted stereoisomer of 19 was subjected to identical
conditions to give substrate 21 (75%). A preliminary analysis
of binding against a panel of bulged oligonucleotides
confirmed that 20 is as effective as other aminoglycosylated
versions of 2 and in some cases is superior to NCSi-gb itself
(Table 1).9 The need for right-handed twist in order to satisfy
molecular recognition in the bulged environment is evident
by comparison to stereoisomer 21 (Table 1, entries 2-4).
The results confirm that a hydrogen bonding group is not
required at the spiro junction, greatly simplifying synthetic
In summary, a versatile and efficient route to mimics of
the natural product metabolite NCSi-gb has been developed.
The lead molecule, available in eight steps from a readily
available template, shows submicromolar and selective
binding to bulged DNA targets and induces slippage at
relevant concentrations, allowing for rational drug design to
address this key molecular biology target. Of potential
significance is the synthetic versatility of the vinyl group in
20, which will allow design and assembly of diverse libraries
for screening.12
Supporting Information Available: Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL0400591
(9) Xi, Z.; Jones, G. B.; Qabaja, G.; Wright, J. W.; Johnson, F. S.;
Goldberg, I. H. Org. Lett. 1999, 1, 1375-1377.
(10) Ruggiero, B. L.; Topal, M. D. J. Biol. Chem. 2004, 279, 23088-
23097.
(11) Kappen, L. S.; Xi, Z.; Jones, G. B.; Goldberg, I. H. Biochemistry
2003, 42, 2166-2173.
(12) We thank the NIH for financial support through Grants RO1GM57123
(to GBJ) and R01GM53793(to IHG).
(8) Attemps to isolate the intermediates in this cascade were unsuccessful.
74
Org. Lett., Vol. 7, No. 1, 2005