Combining the best in triplex recognition: Synthesis and nucleic acid binding of a BQQ-neomycin conjugate

Article abstract of DOI:10.1021/ja034241t

Synthesis of a BQQ-neomycin conjugate is reported. The conjugate combines two ligands, one known to intercalate triplexes (BQQ) and another known to bind in the triplex groove (neomycin). The conjugate stabilizes T·A·T, as well as mixed base DNA triplex,

Full text of DOI:10.1021/ja034241t

Published on Web 06/14/2003
Combining the Best in Triplex Recognition: Synthesis and Nucleic Acid
Binding of a BQQ-Neomycin Conjugate
Dev P. Arya,* Liang Xue, and Paul Tennant
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson UniVersity, Clemson, South Carolina 29634
Received January 19, 2003; E-mail: dparya@clemson.edu
Scheme 1a
Recognition of duplex DNA by oligonucleotides (major groove
binders-DNA triple helices) is a promising approach to a chemical
solution for DNA recognition.1 Triple helix formation has been the
focus of considerable interest because of possible applications in
developing new molecular biology tools as well as therapeutic
agents, and because of the possible relevance of H-DNA structures
in biological systems.2-5 Several intercalators as well as various
DNA minor groove ligands have been shown to bind to DNA triple
helices.4,6,7 Intercalators usually stabilize to a greater extent triple
helices containing T‚A‚T triplets, whereas minor groove binders
usually destabilize triplexes.8 In our quest for new ligands for triple
helix stabilization, we have investigated aminoglycoside antibiotics
(neomycin, Scheme 1).9-11
The stabilization of the poly(dA)‚2poly(dT), poly(rA)‚2poly(rU),9
a mixed base DNA 22mer,11 and DNA/RNA hybrid duplexes/triple
helices10 by neomycin has recently been reported by us. Among
all of the aminoglycosides, neomycin was shown to be the most
effective triplex groove binder that remarkably stabilized DNA and
RNA triple helices with little effect on the double helices. This
a Reagents and conditions: (a) (Boc)2O, DMF, H2O, Et3N, 60 °C, 5 h,
60%; (b) 2,4,6-triisopropylbenzenesulfonyl chloride, pyridine, room tem-
perature, 40 h, 50%; (c) HSCH2CH2NH2‚HCl, NaOEt/EtOH, room tem-
work has complemented the success in the development of triplex
specific intercalators in the past decade, among which the penta-
cyclic BQQ-based structures have been presented as one of the
best in triplex binding selectivity and potency.7,12 We have recently
reported that a pyrene-neomycin conjugate can stabilize poly(dA)‚
2poly(dT) triplex much more effectively and specifically than
neomycin or pyrene at low concentrations.13 Because pyrene by
itself is not a potent intercalator (but a transient one), we
hypothesized that by replacing pyrene with a more specific triplex
binding intercalator such as BQQ,7 new classes of molecules with
high triplex affinity can be identified. A BQQ-neomycin conjugate
has therefore been synthesized by forming a thiourea linkage
between neomycin and benzo[f]quino[3,4-b]quinoxaline derivative
(BQQ) (Scheme 1). We report that the BQQ-neomycin conjugate
is more potent in stabilizing DNA triple helices than neomycin,
BQQ, or a combination of both.
The following assumptions were made in the design of the
conjugate: The amino groups on rings I, II, and IV of neomycin
are necessary in stabilizing and in recognizing the triplex groove
(aminoglycosides without any of these amines do not stabilize
triplexes as efficiently).11 The 5′′-OH on ring III was thus chosen
to provide the linkage to the intercalating unit. The intercalator
(Scheme 1, BQQ-amine12) was linked to neomycin isothiocyanate
3 (prepared in four steps from neomycin)14 in the presence of
4-(dimethylamino)pyridine (DMAP) as a catalyst. The synthesis
rests on the selective conversion of ring III 5′′-OH of neomycin
into a good leaving group (TIBS-2,4,6-triisopropylbenzenesulfonyl)
as previously reported by Tor.15 The displacement of TIBS by
aminoethanethiol, conversion into isothiocyanate, followed by
coupling with the primary amine in BQQ,12 and deprotection with
HCl gives the target conjugate 4 in good yields (Scheme 1).
perature, 18 h, 50%; (d) 1,1′-thiocarbonyldi-2(1H)-pyridone, CH2Cl2, room
temperature, overnight, 86%; (e) BQQ, DMF, DMAP, room temperature,
7 h, 83%; (f) 4 M HCl/dioxane, HSCH2CH2SH, room temperature, 5 min,
58%.
Figure 1. Competition dialysis of various nucleic acids with 1 µM BQQ-
neomycin. 180 µL of different nucleic acids (75 µM per monomeric unit
of each polymer) was dialyzed with 400 mL of 1 µM BQQ-neomycin
(150 mM KCl, 0.5 mM EDTA, 10 mM sodium cacodylate, pH 6.8) solution
for 72 h. At the end of dialysis, 150 µL of nucleic acid samples was carefully
removed and placed in microfuge tubes, and the samples were taken to a
final concentration of 1% (w/v) sodium dodecyl sulfate (SDS). Each mixture
was allowed to equilibrate for 2 h. Each nucleic acid solution was then
further diluted to an overall volume of 4 mL by BPES buffer. The
concentration of BQQ-neomycin after dialysis was determined by fluo-
rescence (FluoroMax-3).16
To ascertain the selectivity of this novel conjugate, we used the
competition dialysis method developed by Chaires.16 Figure 1 shows
competition dialysis results obtained for various nucleic acids with
BQQ-neomycin. No binding is observed with single-stranded DNA
or RNA. Duplex DNA only weakly binds to BQQ-neomycin as
compared to triplex. BQQ-neomycin binding to triplex DNA is
greater than to duplex DNA or single strands. RNA triplex similarly
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J. AM. CHEM. SOC. 2003, 125, 8070-8071
10.1021/ja034241t CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (A) UV melting profile of a 2:1 strand mixture I (dA22‚2dT22)
at 260 nm with 150 mM KCl, 10 mM sodium cacodylate, 0.5 mM EDTA,
pH 6.8: (a) 4 µM BQQ, (b) 4 µM neomycin, (c) 4 µM neomycin + 4 µM
BQQ, (d) no ligand, (e) 4 µM BQQ-neomycin. (B) CD melting profile
(right) of dA22‚2dT22 at 260 nm in the presence of 150 mM KCl, 10 mM
sodium cacodylate, 0.5 mM EDTA, pH 6.8. dA22 ) 1 µM/stand; dT22 ) 2
µM/stand; [BQQ-neomycin] ) 4 µM.
Figure 3. (Left) UV melting profiles of 22mer triplex II (shown below)
at 260 nm in the presence of 150 mM KCl, 10 mM sodium cacodylate, 0.1
mM EDTA, pH 6.8: (a) no ligand, (b) 8 µM BQQ, (c) 8 µM neomycin,
(d) 8 µM neomycin + 8 µM BQQ, (e) 8 µM BQQ-neomycin, (f) 10 µM
BQQ-neomycin. dR22 ) dY22 ) dT22 ) 1 µM/strand. (Right) A computer
model of BQQ-neomycin docked in a T‚A‚T DNA triplex. dR22: 5′d-
(AAAGGAGGAGAAGAAGAAAAAA)3′. dY22: 3′d(TTTCCTCCTCT-
TCTTCTTTTTT)5′. dX22: 5′d(TTTCCTCCTCTTCTTCTTTTTT)3′.
has a higher affinity for the drug than does RNA duplex, even higher
than the 16S ribosomal RNA A-site, the natural target for
aminoglycosides.
even after the duplex melts. At higher concentrations of the drug
(10 µM), only one transition (73 °C, Tm3f1) is observed (Figure
3f).
Thermal denaturation of complex I (dA22‚2dT22) was then studied
{using a 2:1 pyrimidine (dT22) to purine (dA22) strand ratio} by
UV and CD spectroscopy. Figure 2a shows the UV melting profiles
of this 2:1 strand mixture I in the presence of 4 µM neomycin,
BQQ, and BQQ-neomycin. In the absence of ligand, the melting
profile of the mixture is monophasic, which represents the duplex
dissociation at Tm2f1 ) 49 °C. No triplex transition is observed. In
the presence of 4 µM BQQ-neomycin, a new transition emerges
at ∼80 °C. From UV and CD melts (Figure 2b), this new transition
was identified as the dissociation of triplex dA22‚2dT22 directly to
single strands. In the presence of 4 µM BQQ-neomycin, the duplex
melts at 49 °C, and the triplex melts at 80 °C, as was previously
observed with a few other triplex specific intercalators (coralyne).17
A negative CD (260 nm) transition at 47 °C represents duplex
dissociation, while a positive transition at 80 °C represents triplex
dissociation. A similar melting profile is obtained when dA22 and
dT22 are present in a 1:1 ratio, suggesting that disproportionation
of some of the duplex to the triplex occurs in the presence of the
drug.
An ITC titration of the drug binding to dA22‚2dT22 triplex (300
mM NaCl,18 Supporting Information) yields an isotherm that can
be fit to a two binding site model, with one of the Kds’s showing
nanomolar drug binding (27 ( 2 nM). A model depicting the
possible binding of the conjugate to a T‚A‚T triplex is shown in
Figure 3. As BQQ stacks between the triplex bases, neomycin stays
bound in the larger triplex W-H groove. The design of such dual
recognition ligands then opens a new paradigm for recognition of
triplex nucleic acids (DNA, RNA, or hybrid) and should aid in the
development of even more selective and potent conjugates.
Acknowledgment. This work was supported by a NSF-
CAREER award to D.P.A. (CHE/MCB-0134932).
Supporting Information Available: UV melts, scans, and synthesis/
characterization of the conjugate (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
References
Additionally, the transition at 80 °C shows pronounced hysteresis
(indicative of slow triplex annealing), whereas the duplex melting
and annealing transitions overlap (Supporting Information). This
suggests that as little as 4 µM of the ligand leads to a ∆Tm of >60
°C (assuming a triplex Tm of <10 °C in the absence of ligand).
BQQ alone shows triplex melt at 65 °C, whereas neomycin is unable
to induce the triplex under these low concentrations. At higher
BQQ-neomycin concentrations, duplex and triplex transitions
merge to a Tm3f1 transition.
(1) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215-2235.
(2) Kool, E. T. Acc. Chem. Res. 1998, 31, 502-510.
(3) Frank-Kamenetskii, M. D.; Mirkin, S. M. Annu. ReV. Biochem. 1995, 64,
65-95.
(4) Ganesh, K. N.; Kumar, V. A.; Barawkar, D. A. In Supramolecular Control
of Structure and ReactiVity; Hamilton, A. D., Ed.; John Wiley & Sons
Ltd.: New York, 1996; pp 263-327.
(5) Feigon, J.; Wang, E. In Nucleic Acid Structure, 1st ed.; Neidle, S., Ed.;
Oxford University Press: Oxford, 1999; Vol. 1, pp 355-388.
(6) Shafer, R. H. Prog. Nucleic Acid Res. Mol. Biol. 1998, 59, 55-94.
(7) Escude, C.; Nguyen, C. H.; Kukreti, S.; Janin, Y.; Sun, J.-S.; Bisagni, E.;
Garestier, T.; Helene, C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3591-
3596.
The UV melting of a mixed base triplex II (Figure 3) was then
monitored at 260, 280, and 284 nm. Melting curves of triplex II in
the presence of different ligands (BQQ, BQQ-neomycin, and
neomycin) at 8 µM concentration are shown in Figure 3. The
melting profiles are clearly biphasic, as is typically observed with
this triplex.11 Without any ligand, the triplex melts at 30 °C, and
the duplex melts at 56 °C. In the presence of 8 µM BQQ (Figure
3b,d), a broad triplex melt is observed from 32 °C upward.
In the presence of 8 µM BQQ-neomycin, the melting profile
exhibits a broad transition before the duplex, but a much sharper
transition after the duplex melt (Tm3f1 ) 71 °C), with the duplex
transition at 56 °C (similar to what is observed with dA22‚2dT22
triplex). BQQ-neomycin stabilizes T‚A‚T as well as C‚G‚C+ mixed
base triplex DNAs with drug induced triplex stabilization occurring
(8) Lane, A.; Jenkins, T. Curr. Org. Chem. 2001, 5, 845-869.
(9) Arya, D. P.; Coffee, R. L., Jr. Bioorg. Med. Chem. Lett. 2000, 10, 1897-
1899.
(10) Arya, D. P.; Coffee, R. L., Jr.; Charles, I. J. Am. Chem. Soc. 2001, 123,
11093-11094.
(11) Arya, D. P.; Coffee, R. L., Jr.; Willis, B.; Abramovitch, A. I. J. Am. Chem.
Soc. 2001, 123, 5385-5395.
(12) Zain, R.; Marchand, C.; Sun, J.; Nguyen, C. H.; Bisagni, E.; Garestier,
T.; Helene, C. Chem. Biol. 1999, 6, 771-777.
(13) Xue, L.; Charles, I.; Arya, D. P. J. Chem. Soc., Chem. Commun. 2002,
70-71.
(14) Charles, I.; Xue, L.; Arya, D. P. Bioorg. Med. Chem. Lett. 2002, 12, 1259-
1262.
(15) Michael, K.; Tor, Y. Chem.-Eur. J. 1998, 4, 2091-2098.
(16) Ren, J.; Chaires, J. B. Methods Enzymol. 2001, 340, 99-108.
(17) Polak, M.; Hud, N. Nucleic Acids Res. 2002, 30, 983-992.
(18) dA22‚2dT22 triplex does not form below 300 mM NaCl in the absence of
the drug.
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