Reaching into the major groove of B-DNA: Synthesis and nucleic acid binding of a neomycin-Hoechst 33258 conjugate

Article abstract of DOI:10.1021/ja036742k

Synthesis of a neomycin-Hoechst 33258 conjugate is reported. The conjugate combines the ligands known to selectively bind in the duplex and the triplex groove. The conjugate stabilizes DNA duplex over DNA triplex. The conjugate selectively stabilizes the

Full text of DOI:10.1021/ja036742k

Published on Web 09/19/2003
Reaching into the Major Groove of B-DNA: Synthesis and Nucleic Acid
Binding of a Neomycin-Hoechst 33258 Conjugate
Dev P. Arya* and Bert Willis
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson UniVersity, Clemson, South Carolina 29634
Received June 18, 2003; E-mail: dparya@clemson.edu
Scheme 1 a
Recognition of duplex DNA by small molecules (polyamides,
minor groove binders) and triplex forming oligonucleotides (TFOs,
major groove binders) is a promising approach to a chemical
solution for DNA recognition.1 Several intercalators as well as
various DNA minor groove ligands have been shown to bind to
DNA double and triple helices.2 Among the DNA groove binders,
a Reagents and conditions: (i) (a) 5-trifluoroacetamido-1-pentanol, PPh3,
Hoechst 33258, its derivatives, and conjugates have been extensively
DIAD, dioxane, rt, 2 h, 84%; (b) HCl(g), EtOH, 0 °C, quant.; (ii) (a) 2-(3,4-
diaminophenyl)-6-(1-methyl-4-piperazinyl) benzimidazole, HOAc, reflux,
4 h, 38%; (b) K2CO3 in 5:2 MeOH:H2O, rt, overnight, 94%.
studied for DNA duplex binding with a preference for A‚T rich
sequences.3 We have recently shown neomycin to be a triplex-
specific groove binder.2 Neomycin, an aminoglycoside, had largely
been known to bind different RNA structures.4-8 Recent studies
from our lab have, however, revealed that aminoglycosides (in
particular, neomycin) can bind to other A-form structures.9 The
stabilization of poly(dA)‚2poly(dT) triplex,10 small oligomeric
triplexes,11 DNA‚RNA hybrid duplexes,12 RNA triplex,2 and hybrid
triplexes by neomycin has recently been reported by us. Aminogly-
cosides most likely bind in the major groove of these structures
(much like RNA, as the A-form nucleic acids have a narrower major
groove).9 The B-form duplex has a much larger major groove and
does not provide a good shape complementarity to aminoglycoside
binding. Among all 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.2,11 This work has complemented the success in
development of DNA duplex specific groove binders in the past
few decades, among which netropsin, distamycin, and Hoechst
33258 have been the lead compounds. We wished to investigate if
a molecule such as neomycin can be forced into the B-form DNA
major groove. Another intriguing question in this regard was
whether the binding would be driven by Hoechst 33258 (duplex
selective groove binder) or neomycin (triplex selective groove
binder). Such ligands with minor/major groove recognition are also
promising for development of inhibitors of transcription factors.13
Herein, we report the synthesis and nucleic acid binding of a novel
neomycin-Hoechst 33258 conjugate. The conjugate shows remark-
able stabilization of DNA duplexes and destabilization of the DNA
triplex.
Starting with commercially available p-cyanophenol 1, a Mit-
sunobu-type reaction with protected amino alcohol (N-trifluoro-
acetamido protection of 5-aminopentan-1-ol) allowed for successful
conversion to imidate ester 2, which could be subsequently coupled
with 2-(3,4-diaminophenyl)-6-(1-methyl-4-piperazinyl) benzimida-
zole (prepared in five steps utilizing reported procedures)14-16 to
give the desired Hoechst 33258 linker component 3 necessary for
ultimate conjugate synthesis (Scheme 1) (a similar approach has
been reported by McLaughlin).17,18 Using procedures developed by
Tor19 and our laboratory,20,21 successful preparation of an electro-
philic derivative of neomycin was then accomplished (Scheme 2).
Starting from the natural product neomycin B, which is com-
mercially available as the trisulfate salt, Boc (tert-butoxycarbonyl)
protection of the six amino groups followed by conversion to 2,4,6-
Scheme 2 a
a Reagents and conditions: (i) 1,1′-thiocarbonyldi-2(1H)-pyridone, cat.
DMAP, CH2Cl2, rt, 20 h, 95%; (ii) 3, pyridine, rt, overnight, 72%; (iii) 1:1
CH2Cl2, TFA, rt, 3 h, quant.
triisopropylbenzenesulfonyl derivative and subsequent substitution
by aminoethanethiol gave rise to the protected neomycin amine,19
compound 4. Treatment of 2 with 1,1′-thiocarbonyldi-2(1H)-
pyridone using a catalytic amount of DMAP gave isothiocyanate21,22
derivative 5, which was coupled with bis(benzimidazole) 3 and
deprotected to give conjugate 7.
The thermal stability of DNA triple and double helices in the
presence of neomycin, Hoechst 33258, and neomycin-Hoechst
33258 conjugate 7 was investigated using thermal denaturation
monitored by UV absorbance. It was found that 7 displays a marked
effect on the stability of poly(dA)‚poly(dT) duplex when compared
to both neomycin (which is known to have no effect on the thermal
stability of duplex DNA) and Hoechst 33258, which displayed some
degree of stabilization of duplex DNA (Figure 1, left).
In the absence of ligand, the melting profile of poly(dA)‚2poly-
(dT) is biphasic with Tm3f2 ) 34 °C and Tm2f1 ) 72 °C. As
depicted in Figure 1 (left), the dissociation of duplex DNA in the
presence of 7 occurs at a higher temperature (>95 °C) than that of
DNA in the presence of Hoechst 33258 (86 °C) and neomycin (72
°C, unchanged when compared to native duplex melting), suggest-
ing that 7 stabilizes the duplex better than the individual parent
9
12398
J. AM. CHEM. SOC. 2003, 125, 12398-12399
10.1021/ja036742k CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. (Left) UV melting profile of poly(dA)‚2poly(dT) in the presence
of (a) no ligand, (b) 2 µM neomycin, (c) 2 µM Hoechst 33258, (d) 2 µM
neomycin + 2 µM Hoechst 33258, and (e) 2 µM 7. Samples of DNA (15
µM/base triplet) in buffer (10 mM sodium cacodylate, 0.5 mM EDTA, 150
mM KCl, pH 7.2) containing ligand were analyzed for UV absorbance at
260 nm from 20 to 95 °C using a temperature gradient of 0.2 °C/min. (Right)
UV melting profile of d(CGCAAATTTGCG)2 in the presence of (a) no
ligand, (b) 1 µM neomycin, (c) 1 µM Hoechst 33258, (d) 1 µM neomycin
+ 1 µM Hoechst 33258, and (e) 1 µM 7. Samples of DNA (1 µM/duplex)
in BPES buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, 185 mM
NaCl, pH 7.0) containing ligand (1 µM) were analyzed for UV absorbance
at 260 nm from 20 to 95 °C using a temperature gradient of 0.2 °C/min.
Figure 2. (a) (Left) Bar graph of ∆Tm for 22-mer duplexes in the presence
of 4 µM Hoechst 33258 and 4 µM neomycin-Hoechst 33258 7 obtained
from UV melting profiles (solution conditions were identical to those in
Figure 1b). (b) (Right) Computer model of neomycin-Hoechst 33258 (7)
(yellow, linker atoms shown in white) docked in the DNA major-minor
grooves.
to target a structure of preference and should aid in the development
of even more selective and potent conjugates. Development of such
dual-recognition ligands is being further explored in our laboratories
and will be reported in due course.
compounds. Samples containing both neomycin and Hoechst 33258
displayed no difference in Tm from that observed with the individual
molecules. It is important to note that triplex melting was not
observed for poly(dA)‚2poly(dT) in the presence of 7, suggesting
that drug binding prevents the third strand polypyrimidine from
binding in the major groove.
A comparison was then made with a self-complementary DNA
duplex d(CGCAAATTTGCG)2 well-known for Hoechst 33258
affinity.3 UV melting showed increased stability of the duplex in
the presence of 7, with a ∆Tm ) 25 °C (Figure 1 (right)), compared
to ∆Tm ) 14 °C for Hoechst 33258.3
Further studies of numerous duplex DNA 22-mers of varying
G/C content (breaking up stretches of A/T base pairs) were carried
out (please see Supporting Information for the sequences). In all
cases where stretches of at least four A/T base pairs were present,
∆Tm for 7 was at least 10 °C higher than that for Hoechst 33258.
Duplex stabilization by 7 follows the selectivity shown by Hoechst
33258 (Figure 2a), whereas neomycin has no effect on the
stabilization of any duplex. Hoechst 33258 is well-known to have
a primary preference for A/T stretches as low as four base pairs,
suggesting that the binding-induced thermal stabilization by 7 is
largely controlled by the Hoechst 33258 moiety’s ability to bind
to its required stretch of A/T base pairs.
Acknowledgment. This work was supported by a NSF-
CAREER award to D.P.A (CHE/MCB-0134792).
Supporting Information Available: UV scans/melts, synthesis/
characterization of conjugate 7 (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
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A model depicting the possible binding of 7 to a 12-mer duplex
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observed between Hoechst 33258 and duplex DNA3 (∼108 M-1
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as opposed to neomycin binding to triplex (105-106 M-1).11
Conjugates of different linker sizes can then perhaps be designed
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