Synthesis and NMR binding study of a chiral spirocyclic helical analogue of a natural DNA bulge binder

Article abstract of DOI:10.1021/ol048152c

(Chemical Equation Presented) Synthesis of chiral spirocyclic helical compounds which mimic the molecular architecture of the potent DNA bulge binder obtained from the antitumor agent NCS-chrom has been accomplished. Structural analysis of the compounds b

Full text of DOI:10.1021/ol048152c

ORGANIC
LETTERS
2
004
Vol. 6, No. 26
833-4836
Synthesis and NMR Binding Study of a
Chiral Spirocyclic Helical Analogue of a
Natural DNA Bulge Binder
4
Nilesh W. Gaikwad, Geum-Sook Hwang, and Irving H. Goldberg*
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical
School, Boston, Massachusetts 02115
irVing_goldberg@hms.harVard.edu
Received September 13, 2004 (Revised Manuscript Received November 15, 2004)
ABSTRACT
Synthesis of chiral spirocyclic helical compounds which mimic the molecular architecture of the potent DNA bulge binder obtained from the
antitumor agent NCS-chrom has been accomplished. Structural analysis of the compounds by CD and NMR is presented. NMR titration study
indicates binding of P,r-helimer (1d) at a two-base bulge site in a DNA oligomer, providing insight to the design of agents as specific probes
of a bulged structure in nucleic acids.
The helix represents an important example of conformational
chirality that is ubiquitous in the structure of biomolecules
and is also observed in small molecules. The conformational
therapeutic potential, as DNA-bulged structures have been
shown to have significant biological roles. Unique helix-
3
1
shaped molecular geometry that fits tightly in the triangular
prism binding pocket formed by the two looped out bases at
the bulge site was found to be responsible for the activity of
1. The orientation of ring systems, transposed approximately
60° by the spirolactone, and the right-handed ∼35° twist
asymmetry in the host and/or guest molecule plays an
important role in molecular recognition. One such example
that we recently studied in our laboratory is wedge-shaped
spirolactone 1, formed in the base-catalyzed cyclo-aroma-
tization of the neocarzinostatin chromophore (NCS-chrom),
a potent antitumor antibiotic that has been shown to have
very high and specific affinity for DNA containing a two-
4
renders the molecule, 1, a helical shape. On the basis of
spectroscopic and molecular modeling studies, it was con-
cluded that the key features required for specific bulge
2
nucleotide bulge. The prospect of selective recognition of
a DNA bulge by a small molecule could have important
(2) (a) Kappen, L. S.; Goldberg, I. H. Biochemistry 1993, 32, 13138.
b) Hensen, O. D.; Chin, D.-H.; Stassinopoulos, A.; Zink, D. L.; Kappen,
(
L. S.; Goldberg, I. H. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4534. (c) Xi,
Z.; Mao, Q. K.; Goldberg, I. H. Biochemistry 1999, 38, 4342. (d) Yang, C.
F.; Jackson, P. J.; Xi, Z.; Goldberg, I. H. Bioorg. Med. Chem. 2002, 10,
1329.
(3) (a) Kunkel, T. A. Nature 1993, 365, 207. (b) Lilley, D. M. J. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 7140. (c) Payet, D.; Hillish, A.; Lowe,
N.; Diekmann, S.; Travers, A. J. Mol. Biol. 1999, 294, 79. (d) Degtyareva,
N.; Subramanian, D.; Griffith, J. D. J. Biol. Chem. 2001, 276, 8778.
(4) Stassinopoulos, A.; Ji, J.; Gao, X.; Goldberg, I. H. Science 1996,
272, 1943.
(
1) (a) Furche, F.; Ahlrichs, R.; Wachsmann, C.; Weber, E.; Sobanski,
A.; Voegtle, F.; Grimme, S. J. Am. Chem. Soc. 2000, 122, 1717. (b)
Theilacker, W.; Boehn, H. Angew. Chem., Int. Ed. Engl. 1967, 6, 251. (c)
Rappoport, Z.; Biali, S. E. Acc. Chem. Res. 1997, 30, 307. (d) Kiupel, B.;
Niederalt, C.; Nieger, M.; Grimme, S.; Vogtle, F. Angew. Chem., Int. Ed.
1
998, 37, 3031. (e) Djukic, J.-P.; Michon, C.; Maisse-Francois, A.;
Allagapen, R.; Pfeffer, M.; Dotz, K. H.; De Cian, A.; Fischer, J. Chem.
Eur. J. 2000, 6, 1064. (f) Grimme, S.; Harren, J.; Sobanski, A.; Voegtle, F.
Eur. J. Org. Chem. 1998, 1491.
1
0.1021/ol048152c CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/25/2004

Scheme 1. Synthesis of Spirocyclic Diketo 13-Endo Alcohol
Aglycon 6
Scheme 2. Glycosylation of Aglycon 6 with Acetyl-Protected
Aminoglycoside
3
acetyl deprotection using NaOMe, Et N, and Mg/MeOH were
unsuccessful (Scheme 2).
The probable reason is thought to be that the deacetylation
reaction conditions catalyze the retroaldol-type ring opening
(
Scheme 3).
binding are (1) two independent aromatic π systems, (2) a
spirocyclic ring junction capable of offsetting the systems
by 30-40°, and (3) a pendant aminosugar moiety to enhance
binding to the sugar phosphate backbone at the bulged site.
In our attempts to design and synthesize structural mimics
Scheme 3. Proposed Retroaldol-Type Ring-Opening
4
Mechanism
of 1 with the potential of therapeutic use, we previously
5
prepared a number of spirocyclic compounds. Fluorescence
binding and NMR structural studies showed that these
compounds, unlike 1 which is a major groove binder, bind
through the minor groove and are approximately 10 times
less potent and selective toward the bulges.5 The apparent
reason for lower affinity is that the aminoglycoside moiety,
unlike natural product 1, is attached to C-25 of aglycon by
a â-linkage rather than at C-13 by an R-glycoside linkage.
In this regard, we pursued the synthesis of a stable structural
mimic of 1, a diketospirocyclic glycoside with a right-handed
helix and sugar moiety attached at C-13 by R-glycoside
linkage 1d.
,6
To overcome this problem, we chose easily available
benzyl-protected glycoside 9. Trichloroacetimidate 10, syn-
thesized from 9, was coupled with 6 in good yields. The
global benzyl deprotection was easily carried out by reductive
Diels-Alder product 2, synthesized as described previ-
Pd/C8
cleavage of the benzyl protecting groups using H
2
5
ously, was reduced with NaBH
4
, and the resulting alcohol
(
Scheme 4). The crude products were purified on silica gel
was protected with TBSOTf to yield 3. Oxidation of the
double bond followed by base-catalyzed spiro-aldol cycliza-
tion gave hydroxyketone 4 in good yield, isolated as a
mixture of the endo isomer accompanied by ∼35% of its
exo isomer. The endo isomer was oxidized and further
deprotected to yield spirocyclic diketo endo-alcohol, 5. In a
modified Mitsunobu reaction 5 was esterified with picolinic
acid, and the resulting ester was cleaved under essentially
Scheme 4. Glycosylation of Aglycon 6 Using
Benzyl-Protected Glycoside
2
neutral conditions using Cu(OAc) and methanol to yield
7
the desired spirocyclic diketo exo-alcohol 6 (Scheme 1).
Trichloroacetimidate 7, obtained from the aminoglucoside,
was coupled with 6, and the products were subjected to Fmoc
deprotection to give mixture of diastereomers 8 (data not
shown). Unfortunately, all the attempts of further global
and by reversed-phase HPLC. HPLC chromatography indi-
(
5) (a) Xi, Z.; Jones, G. B.; Qabaja, G.; Wright, J.; Johnson, F. S.;
1
Goldberg, I. H. Org. Lett. 1999, 1, 1375. (b) 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.
cated the presence of two peaks in a ∼70:30 ratio. H NMR
analysis of the separated peaks indicated the presence of a
(
6) (a) Hwang, G.-S.; Jones, G. B.; Goldberg, I. H. Biochemistry 2002,
4
2
2, 8472. (b) Hwang, G.-S.; Jones, G. B.; Goldberg, I. H. Biochemistry
004, 43, 641.
(8) Myers, A. G.; Glatthar, R.; Hammond, M.; Harrington, P. M.; Kuo,
E. Y.; Liang, J.; Schaus, S. E.; Wu, Y.; Xiang, J.-N. J. Am. Chem. Soc.
2002, 124, 5380.
(7) Sammakia, T.; Jacobs, J. S. Tetrahedron Lett. 1999, 40, 2685-2688.
4834
Org. Lett., Vol. 6, No. 26, 2004

Figure 2. CD spectra of 1a-d.
aglycon part of the helimers was determined by circular
dichroism (CD) spectroscopy (Figure 2).
Figure 1. Energy-minimized (mm2) molecular structures of 1a-
d.
The CD spectrum of 1a-d in acetonitrile displayed
absorptions at 209, 235, and 280 (weak). Accordingly, the
CD of 1a and 1c in acetonitrile showed a positive cotton
effect (CE) at 209, 280 and a negative CE at 235. 1b and
1
d showed positive CE at 235 and negative CE at 209, 280,
mixture that was further purified by chiral HPLC to yield
two sets of helimers 1a, 1b and 1c, 1d (Figure 1).
Structural assignments of these helimers were made using
which is complementary to 1a and 1c. All four helimers
showed crossover at 223 and 269 nm. The peaks at 209,
2
π to π* transitions in the UV spectra. The positive CD
spectra for 1b and 1d suggests the helix with right-
handedness and hence P conformation, while 1a and 1c, with
9
10
11
12
35, and 280 nm are CEs associated with the corresponding
2D NMR.
Chemical shifts of anomeric proton of 1a, 1b, 1c, and 1d
were 4.54 (d, J ) 7.0 Hz), 4.66 (d, J ) 7.5 Hz), 5.09 (d, J
3.78 Hz), and 5.19 (d, J ) 3.66 Hz) ppm, respectively.
)
1
left-handedness has M configuration. On the basis of this
NMR data for 1a and 1b are characteristic of â-anomeric
protons and for 1c and 1d are of R. The handedness of the
analysis, it was confirmed that 1d has a right-handed aglycon
helix and the glucopyran is attached to it by an R-glycoside
linkage, indicating that 1d is an exact structural mimic of
NCSi-gb, 1. Therefore, we used 1d for further binding studies
with bulged DNA.
1
(
9) 1a: H NMR (CD3CN) δ 8.04 (s, 1H, H-15), 7.94 (d, 1H, J ) 8 Hz,
H-17), 7.45-7.53 (m, 3H, H-18,19,20), 7.33-7.38 (m, 2H, H-3,4), 6.99 (t,
H, H-2), 6.46 (d, 1H, J ) 7 Hz, H-1), 6.21 (s, 1H, H-22), 5.39 (s, 1H,
H-13), 4.81 (d, 1H, J ) 7 Hz, H-11), 4.53 (d, 1H, J ) 7 Hz, H-1′), 3.95 (d,
H, J ) 9 Hz, H-6′), 3.78 (dd, 1H, J ) 4.5, 15.5 Hz, H-6′), 3.55 (m, 2H,
H-6b,12), 3.45 (m, 1H, H-5′), 3.36 (m, 2H, H-3′,4′), 3.23 (m, 2H, H-2′,6a),
.07 (m, 2H, H-7b,24b), 2.72 (ddd, 1H, J ) 5.5, 6, 6.5 Hz, H-7a), 3.43
1
Since 1d is weakly fluorescent, its binding to the two-
base bulge-containing DNA oligomer (Figure 3) was better
1
3
1
3
(11) 1c: 1H NMR (CD3CN) 7.97 (s, 1H, H-15), 7.87 (d, 1H, J ) 8.2
Hz, H-17), 7.45 (m, 2H, H-18,20), 7.40 (t, 1H, J ) 7.88 Hz, H-3), 7.34 (t,
1H, J ) 7.41 Hz, H-19), 7.29 (d, 1H, J ) 7.88 Hz, H-4), 6.97 (t, 1H, J )
7.73 Hz, H-2), 6.43 (d, 1H, J ) 7.57 Hz, H-1), 6.07 (s, 1H, H-22), 5.20 (s,
1H, H-13), 5.09 (d, 1H, J ) 3.78 Hz, H-1′), 4.77 (d, 1H, J ) 7.25 Hz,
H-11), 3.83 (bd, 1H, J ) 8.83 Hz, H-6′), 3.72 (bd, 2H, J ) 8.51 Hz, H-6′,-
12), 3.61 (m, 1H, H-3′), 3.50 (m, 2H, H-2′,5′), 3.31 (m, 2H, H-4′,6b), 3.17
(m, 1H, H-6a), 2.99 (m, 2H, H-7b, 24b), 2.65 (m, 1H, H-7a), 2.30 (dd, 1H,
J ) 8.51, 19.0 Hz, H-24a); MS calcd 553.1838, obsd 553.1.
(
dd, 1H, J ) 8, 19 Hz, H-24a); CNMR (CD3CN) δ 215.76, 210.93, 147.74,
1
1
7
5
40.26, 137.59, 135.59, 131.82, 130.53, 128.74, 127.97, 127.82, 127.76,
27.55, 126.28, 126.14, 125.92, 125.75, 125.72, 100.91, 78.09, 76.58, 76.32,
1.78, 70.53, 61.85, 52.53, 45.49, 42.49, 37.86, 27.33, 24.52; MS calcd
53.1838, obsd 553.1878.
(
1
10) 1b: H NMR (CD3CN) δ 8.05 (s, 1H, H-15), 7.91 (d, 1H, J ) 8.5
Hz, H-17), 7.52-7.39 (m, 4H, H-3,18,19,20), 7.32 (d, 1H, J ) 8 Hz, H-4),
.02 (t, 1H, J ) 7.5 Hz, H-2), 6.48 (d, 1H, J ) 8 Hz, H-1), 6.03 (s, 1H,
H-22), 5.39 (s, 1H, H-13), 4.79 (d, 1H, J ) 7 Hz, H-11), 4.66 (d, 1H, J )
.5 Hz, H-1′), 3.86 (d, 1H, J ) 10.5 Hz, H-6′), 3.69 (dd, 1H, J ) 5.75, 15
7
1
7
(12) 1d: H NMR (CD3CN) δ 7.99 (s, 1H, H-15), 7.87 (d, 1H, J ) 8.24
Hz, H-6′), 3.49-3.58 (m, 2H, H-6b,12), 3.38-3.43 (m, 2H, H-4′,5′), 3.31
Hz, H-17), 7.43 (m, 4H, H-3,18,19,20), 7.27 (d, 1H, J ) 8.24 Hz, H-4),
6.99 (t, 1H, J ) 7.62 Hz, H-2), 6.45 (d, 1H, J ) 7.94 Hz, H-1), 5.95 (s,
1H, H-22), 5.25 (s, 1H, H-13), 5.19 (d, 1H, J ) 3.66 Hz, H-1′), 4.79 (d,
1H, J ) 7.02 Hz, H-11), 3.77 (bd, 1H, J ) 11.6 Hz, H-6′), 3.63 (dd, 1H,
J ) 5.49 Hz, H-6′), 3.56 (m, 1H, H-3′), 3.46 (m, 2H, H-5′, 12), 3.35 (m,
2H, H-2′,4′), 3.26 (t, 1H, J ) 9.0 Hz H-6b), 3.19 (m, 1H, H-6a), 3.01 (m,
2H, H-7b, 24b), 2.66 (m, 1H, H-7a), 2.18 (dd, 1H, J ) 10.68, 20 Hz, H-24a);
MS calcd 553.1838, obsd 553.1.
(
t, 1H, J ) 9 Hz, H-2′), 3.23 (ddd, 1H, J ) 1.5, 6, 15.8 Hz, H-6a), 3.12
(
dd, 1H, J ) 8, 9 Hz, H-3′), 3.03-3.08 (m, 2H, H-7b,24b), 2.9 (bs, 1H,
13
OH), 2.67-2.74 (m, 1H, H-7a), 2.28 (dd, 1H, J ) 9, 19.5 Hz, H-24a);
C
NMR (CD3CN) δ 211.38, 208.86, 145.46, 139.87, 138.53, 135.41, 134.62,
1
1
5
30.65, 128.76, 127.94, 127.91, 127.83, 127.68, 127.66, 126.11, 126.08,
25.88, 125.77, 107.79, 77.28, 76.36, 75.57, 73.74, 71.63, 61.06, 55.11,
2.12, 44.06, 37.72, 27.28, 24.55; MS calcd 553.1838, obsd 553.1902.
Org. Lett., Vol. 6, No. 26, 2004
4835

Figure 3. Structure of the bulged DNA oligomer.
monitored by 1D NMR spectroscopy in 10 mM phosphate
1
buffer, pH 8, at 25 °C. The H NMR spectra of the
nonexchangeable protons of 1d, free DNA oligomer, and
1d-DNA complex are given in Figure 4A, B, and C,
respectively. The formation of the 1d-DNA complex is
indicated by the changes in the chemical shifts of several
residues upon addition of the 1d. The effect is most evident
for the methyl and aromatic base protons.
Assignments of the several proton resonances of the free
DNA oligomer and 1d-DNA complex were obtained by
analysis of NOESY and TOCSY spectra. The peaks for the
methyl protons of T8 and T21 that are either bulge residues
or adjacent to them are downfield shifted upon complex
formation, whereas the methyl protons for T16 and T25 that
are residues far from the bulge site remain unchanged in the
chemical shift. In addition, the base proton for A20H8
adjacent to bulge residues is shifted to the downfield region
upon complex formation, as indicated in Figure 4. The
protons of the DNA residues that are subjected to chemical
shift changes by the binding of 1d are similar to those by
the binding of natural product, but the magnitudes of the
chemical shift changes in the 1d-bulged DNA complex are
smaller than those in the natural product-bulge DNA
complex, indicating that 1d binds to the bulge site of DNA
but the induced DNA conformational changes upon complex
formation are different from those of the natural product.
Similar studies with 1a, which has the least favored
conformation, did not show significant chemical shift changes
in 1a-bulged DNA complex (data not shown).
Figure 4. 1H NMR spectra of (A) 1d in CD
DNA oligomer, and (C) the 1d-bulged DNA complex in D
5 °C.
OD, (B) free bulged
O at
3
2
2
shed light on the design of small molecules as specific probes
of bulged structures in nucleic acids.
Acknowledgment. We thank the NIGMS (RO1GM-
0
53793) for generous support of this project.
Supporting Information Available: Experimental mate-
rial and characterization data. This material is available free
of charge via the Internet at http://pubs.acs.org.
In conclusion, novel spirocyclic helimer analogues of
cycloaromatization products of NCS-chrom have been
synthesized. P,R-helimer(1d), which is a structural mimic
of 1, binds to bulged DNA at the bulge site. These results
OL048152C
4836
Org. Lett., Vol. 6, No. 26, 2004