Pyrene-neomycin conjugate: Dual recognition of a DNA triple helix

Article abstract of DOI:10.1039/b108171c

We report the synthesis of pyrene-neomycin conjugate and its ability to stabilize DNA/RNA triple helices.

Full text of DOI:10.1039/b108171c

Pyrene–neomycin conjugate: dual recognition of a DNA triple helix†
Liang Xue, I. Charles and Dev P. Arya*
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University SC 29634, USA.
E-mail: dparya@clemson.edu; Fax: +1 864-656-6613; Tel: +1 864-656-1106
Received (in Columbia, MO, USA) 9th September 2001, Accepted 6th November 2001
First published as an Advance Article on the web 5th December 2001
We report the synthesis of pyrene–neomycin conjugate and
its ability to stabilize DNA/RNA triple helices.
The intercalator (Scheme 1, pyrene) was linked to neomycin
amine 2 (prepared in 3 steps from neomycin) using its activated
acid. A successful example of coupling of pyrene succinimide
ester with neomycin amine 2 is illustrated in Scheme 1. The
synthesis rests on the selective conversion of ring III 5B-OH of
neomycin into a good leaving group, 2,4,6-triisopropylbenzene-
sulfonyl (TIBS), as previously reported by Tor.9–10 The
displacement of the TIBS leaving group by aminothiol,
followed by coupling with the activated ester and deprotection
with HCl gives the target conjugate 3 in good yield (Scheme
1).
The UV melting studies of triplexes formed from poly-
(dA)·poly(dT) and poly(dT) were carried out using UV
spectroscopy at 260 and 284 nm. Melting curves of poly-
(dA)·2poly(dT) with different ligands, pyrene–neomycin, neo-
mycin and 1-aminopyrene at 4mM concentration are shown in
Fig. 1. The melting profiles are clearly biphasic, as typically
observed when polydA·2polydT triplex is formed. The low
temperature transition represents the conversion from the
triplex to duplex and a single-strand of poly(dT). The high
temperature transition represents the dissociation of the duplex,
poly(dA)·poly(dT) into single strands. Without any ligand,
Oligonucleotides directed to a precise DNA sequence provide a
promising approach to artificially control the transcription
process.1,2 Triplex DNA has attracted attention because of the
potential application of triplex forming oligonucleotides
(TFOs) as therapeutic agents, for applications such as intra-
cellular gene targeting, and as rational chemical solutions to
sequence specific recognition of a DNA duplex.1,3
A popular strategy to improve triplex stability involves the
use of intercalators and groove binding ligands.4–7 Most groove
binders prefer duplex grooves or even destabilize triplex
DNA.4–7 There is little information available for antibiotics that
selectively bind DNA triplex grooves or RNA triplex grooves.
The stabilization of the poly(dA)·2poly(dT), a mixed base DNA
22 mer, RNA and hybrid triple helices by neomycin has recently
been reported by us.6–8 Neomycin was shown to be the most
effective triplex groove binder that remarkably stabilized DNA
and RNA triple helices with little effect on DNA double helix.
We hypothesized that with a combination of neomycin’s strong
ionic/groove recognition and an intercalating moiety, new
classes of molecules with high triplex affinity can be identified.
A pyrene–neomycin conjugate has therefore been synthesized
by forming an amide linkage between neomycin B and
1-pyrenebutyric acid N-hydroxysuccinimide ester (Fig. 1). We
report that pyrene–neomycin is more potent in stabilizing DNA
triple helices than neomycin B or pyrene or a combination of
both. It increases the melting temperature of a DNA triple helix
by > 10 °C compared to neomycin B at 4 mM concentration
{base triplet–drug ratio (rdb) = 0.26}.
The following assumptions were made in the design of the
conjugate. (i) The amino groups on rings I, II and IV are
necessary in stabilizing and in recognizing the Watson–
Hoogsteen groove (aminoglycosides without any of these
amines do not stabilize triplexes as efficiently).7 (ii) For triplex
stabilizing structures based on neomycin, these amines must be
retained. The 5B-OH on ring III was thus chosen to provide the
linkage to the intercalating unit.
Fig. 1 UV melting profile of poly(dA)·2poly(dT) at 260 nm in the presence
of 150 mM KCl, 10 mM sodium cacodylate, 0.5 mM EDTA, pH 7.2. (a) No
ligand, (b) 4 mM-neomycin, (c) 4 mM neomycin + 4 mM 1-aminopyrene, (d)
4 mM pyrene–neomycin, (e) 4 mM 1-aminopyrene. [DNA] = 15 mM per
base triplet, Tm = 1 °C. (The y-axis has been artificially offset to
differentiate the melting curves).
† Electronic supplementary information (ESI) available: NMR spectra, UV
spectra, extinction coefficients, melting curves of pyrene–neomycin
conjugate, details of modeling studies. See http://www.rsc.org/suppdata/cc/
b1/b108171c/
Scheme 1 Reagents and conditions a–e: (a) (Boc)2O, DMF, H2O, Et3N, 60 °C, 5h, 60%; (b) 2,4,6-triisopropylbenzenesulfonyl chloride, pyridine, rt, 40 h,
50%; (c) HCl·H2NCH2CH2SH, NaOEt–EtOH, rt, 18h, 50%; (d) 1-pyrenebutyric acid N-hydroxysuccinimide ester, CH2Cl2, DMAP, rt, overnight, 80%; (e)
4 M HCl–dioxane, HSCH2CH2SH, rt, 5 min, 80%.
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This journal is © The Royal Society of Chemistry 2002

Fig. 2 Plot of variation of Tm3?2 and Tm2?1 of poly(dA)·2poly(dT) as a
function of increasing pyrene–neomycin and neomycin concentrations.
Solution conditions: same as Fig. 1.
Fig. 4 Pyrene–neomycin docked in the Watson–Hoogsteen groove of a TAT
DNA triplex. (Atom colors: pyrene–neomycin; purple: poly(dA); white:
poly(dT); green: poly(dT)—the third strand.)
triplex melts at 32 °C. In the presence of 4 mM neomycin and 4
mM pyrene–neomycin, triplex Tm rises to 43 and 58 °C,
respectively. These results show that pyrene–neomycin can
stabilize the triplex much more effectively than neomycin at
these low concentrations (rdb = 0.26). This is quite remarkable
since pyrene itself is not a good triplex stabilizer. Addition of 4
mM 1-aminopyrene and 4 mM neomycin to a DNA triplex leads
to a small DTm3?2 increase (a biphasic transition, Fig. 1c, Fig.
2). Covalent attachment of the two yields a single transition
with a DTm about 10 °C higher than the sum of the two (added
together). With all three ligands, duplex melting points were
unchanged (71°C), signifying that both neomycin and pyrene–
neomycin (at these concentrations) have no effect on the
stabilization of this DNA duplex. At higher pyrene–neomycin
concentrations, a larger increase in Tm is observed, but the
DDTm values (from neomycin) do not increase significantly,
suggesting saturation of the drug binding site at rdb ~ 0.13.
A slightly different picture emerges with the RNA triplex.
DTm plots of poly(rA)·2poly(rU) with different ligands,
1-aminopyrene, pyrene–neomycin, and neomycin from 0–4 mM
concentration are shown in Fig. 3. Without any ligand, Tm3?2
is 32 °C; while in the presence of 4 mM pyrene–neomycin,
Tm3?2 rises to 41 °C and Tm2?1 rises from 49 to 55 °C.
Pyrene–neomycin then stabilizes RNA triplex as well as the
duplex, but the DTm3?2 values are slightly less than those
induced by neomycin. Duplex stabilization by both ligands is,
however, quite comparable. The inability of pyrene to inter-
calate/stabilize an RNA triplex is perhaps responsible for this
lower stabilization observed with the conjugate (1-aminopyrene
has no effect on the RNA duplex/triplex, Fig. 3). Appropriately
designed conjugates could then, in principle, be made to
differentiate such higher order DNA/RNA nucleic acid struc-
tures, and help us explore the structural variations responsible
for their recognition.
Computer modeling of pyrene–neomycin docked in a TAT
triplex suggests that pyrene can intercalate between the base
pairs while neomycin stays bound to the Watson–Hoogsteen
groove (Fig. 4). Ring I may be embedded inside the groove with
amino groups H-bonded to the anti-parallel T strand, whereas
the ribose can provide the optimum conformation and size for a
good fit into the triplex groove. Rings I and IV are responsible
for bringing the two pyrimidine strands together. Because of
this possible dual binding mode of triplex recognition, pyrene–
neomycin conjugate shows significant ability to stabilize DNA
triple helices. Such chromophores should then be effective
model ligands for surveying the groove recognition properties
of a triplex bound neomycin. These results are now being
extended to the stabilization of mixed sequences/hybrid struc-
tures and will be reported in due course.
Financial support for the work was provided by Clemson
University and Greenville Hospital System. The support of
NMR facility by NSF grant (CHE-9700278) is gratefully
acknowledged. We thank Dr Ljiljana Micovic for help with
modeling studies.
Notes and references
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Fig. 3 Plot of variation of Tm3?2 and Tm2?1 of poly(rA)·2poly(rU) as a
function of pyrene–neomycin and neomycin. 10 mM sodium cacodylate,
0.1 mM EDTA, pH 6.8. (a) Triplex + neomycin, (b) triplex + pyrene–
neomycin, (c) duplex + pyrene–neomycin, (d) duplex + neomycin, (e)
triplex + 1-aminopyrene, (f) duplex + 1-aminopyrene. [RNA] = 30 mM
base triplet.
10 S. R. Kirk, N. W. Luedtke and Y. Tor, J. Am. Chem. Soc., 2000, 122,
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