Triple recognition of B-DNA

Article abstract of DOI:10.1016/j.bmcl.2009.07.079

A novel conjugate of Hoechst 33258, pyrene and neomycin was synthesized and examined for its binding and stabilization of A-T rich DNA duplexes using spectroscopic and viscometric techniques. The conjugate, containing three well known ligands that bind nucleic acids albeit in different binding modes, was found to significantly stabilize DNA over parent conjugates containing only one or both of the other recognition elements. The study represents the first example of DNA molecular recognition capable of minor/major groove recognition in conjunction with intercalation.

Full text of DOI:10.1016/j.bmcl.2009.07.079

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4974–4979
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triple recognition of B-DNA
*
Bert Willis, Dev P. Arya
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel conjugate of Hoechst 33258, pyrene and neomycin was synthesized and examined for its binding
and stabilization of A-T rich DNA duplexes using spectroscopic and viscometric techniques. The conju-
gate, containing three well known ligands that bind nucleic acids albeit in different binding modes,
was found to significantly stabilize DNA over parent conjugates containing only one or both of the other
recognition elements. The study represents the first example of DNA molecular recognition capable of
minor/major groove recognition in conjunction with intercalation.
Received 11 July 2009
Accepted 14 July 2009
Available online 19 July 2009
Keywords:
Neomycin
DNA recognition
Intercalation
Minor groove
Major groove
Ó 2009 Elsevier Ltd. All rights reserved.
Molecular recognition of nucleic acids is an important endeavor
for our understanding of biological processes. A better understand-
ing of nucleic acid recognition may ultimately provide us with the
ability to regulate cellular events. For example, a better under-
standing of DNA shape recognition (A, B or Z-forms) is crucial for
defining the principles for development of gene-regulatory drugs.
B-Form DNA recognition is traditionally accomplished through
the minor groove, major groove or stacking between the base pairs
(intercalation). A number of natural products have been shown to
possess structures characteristic of both groove-binding and inter-
calation.1,2 Selected examples include rebeccamycin (indolocar-
bazole family), nogalamycin (anthracycline family), altromycin
(pluramycin family), and mithramycin (aureolic acid family; see
Supplementary data for chemical structures). Furthermore, quite
a few natural products have been shown to display multiple DNA
binding modes. For example, nogalamycin has been3 reported to
thread DNA, exhibiting intercalation by the central chromophore,
with saccharide regions extending from both ends to bind both
major and minor grooves.3
We have reported on the design of molecules that recognize
DNA using simultaneous recognition of the minor and the major
groove.4–8 Previous attempts have been made to design molecules
that recognize DNA using intercalation and minor groove binding
simultaneously.1,9,10 No successful attempts have been made to
combine the three recognition motifs, to the best of our
knowledge. This report describes one such endeavor in the prepa-
ration and DNA-binding analysis of a novel neomycin–Hoechst
33258–pyrene conjugate, termed ‘NHP’. The goal of this work
was to establish a general multi-recognition scaffold utilizing three
well-established DNA binders. Once a ‘proof of concept’ approach
is deemed feasible, as shown in this manuscript, ligand based tri-
ple-recognition of biologically-relevant DNA sequences could be
addressed.
Recently, neomycin, an aminoglycoside antibiotic, has been
shown to be a versatile scaffold in probing nucleic acid recogni-
tion.7,11 From its well known ability to bind RNA and recent reports
of DNA triplex and nucleic acid hybrid stabilization,12 neomycin
has since been shown to stabilize both A-form (or A-like)5 and B-
form4,11 nucleic acid structures via conjugation with such ligands
as intercalators (pyrene,13 BQQ,14 ethidium15,16) or groove binders
(Hoechst 33258,4,11 nucleic acids.6,17,18) Herein, we expand the
recognition of DNA using these multi recognition motifs. A neomy-
cin–Hoechst 33258–pyrene conjugate, ‘NHP’, was synthesized.
Conjugates of neomycin and pyrene, Hoechst 33258 and pyrene,
and neomycin and Hoechst 33258 (‘NP’, ‘HPA’, and ‘NH’, respec-
tively), were also synthesized and utilized as controls in these
experiments. All structures of the conjugates used in the study
are shown in Figure 1.
UV melting experiments exhibit the increased stabilization of
DNA by NHP. Tm enhancements by individual recognition elements
were resolved using control conjugates containing only one or both
of the other recognition elements. Fluorescence techniques indi-
cate enhanced binding over parent conjugates, with a binding site
size larger than that of Hoechst 33258. Induced Circular Dichroism
studies were used to identify the groove binding of Hoechst 33258
and intercalation of pyrene into DNA. Classical viscometric tech-
niques also detail the binding mode of the pyrene moiety, support-
ing the intercalative nature of pyrene in NHP.
* Corresponding author.
E-mail address: dparya@clemson.edu (D.P. Arya).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.079

B. Willis, D. P. Arya / Bioorg. Med. Chem. Lett. 19 (2009) 4974–4979
4975
H
N
HN
N
N
N
N
O
O
NH2
O
O
HO
HO
H2N
N
O
O
H
O
S
O
NH2
O
H
S
OH
N
H
N
H
O
NH2
OH
O
OH
O
"NH"
H2N
HO
NH2
O
HO
HO
H2N
N
H
H
O
S
O
NH2
H
N
O
S
OH
N
N
H
OH
O
O
H
NH2
O
O
OH
O
"NP"
H2N
HO
H
N
HN
N
N
N
N
O
O
O
O
H
N
O
N
HN
NH2
O
N
N
N
N
"HPA"
O
NH2
O
O
O
O
HO
HO
H2N
N
H
H
O
S
O
NH2
O
S
OH
N
N
N
H
O
O
H
NH2
OH
O
OH
O
"NHP"
H2N
HO
Figure 1. Structures of novel conjugates used in this study.
To synthesize the target conjugate (NHP), the Hoechst–pyr-
ene–amine portion was first prepared (Scheme 1). Monotosylate
5 was coupled with p-cyanophenol using Mitsunobu conditions
to afford 6 in good yields. Tosylate substitution with mono-pro-
tected diamine 7 under basic conditions provided intermediate
8. Reaction of the secondary amine of 8 with pyrene succini-
mide ester 9, followed by standard coupling with 11 in an
ethanol/acetic acid mixture provided the desired Hoechst–pyr-
ene–amine (HPA, 3) after deprotection (K2CO3, MeOH/H2O) of
the terminal amine. Reaction of 3 with neomycin isothiocyanate
12, followed by Boc deprotection of coupled product 13 gives
the desired triple recognition agent 4 (Scheme 2) as a trifluoro-
acetate salt.
To test the effect of the conjugates on oligomeric DNA duplex
stability, UV melting profiles of dA22ꢀdT22 in the absence and
presence of various ligands were gathered. The resulting Tm
associated with each DNA complex is listed in Figure 2 (right).
A much more significant and noticeable shift in the Tm for

4976
B. Willis, D. P. Arya / Bioorg. Med. Chem. Lett. 19 (2009) 4974–4979
CN
NC
OH
HO
O
O
OTs
i
5
O
O
O
OTs
6
ii
CN
O
NHCOCF3
H2N
O
7
O
O
O
NHCOCF3
N
O
N
O
H
O
8
N
O
O
O
iii
11
N
O
CN
O
9
N
HN
O
NHCOCF3
O
N
O
NH2
NH2
O
10
N
N
iv
diamine 11
N
HN
N
HN
O
O
N
O
O
O
O
NH2
3 "HPA"
Scheme 1. Preparation of Hoechst 33258–pyrene–amine. Reagents and conditions: (i) p-cyanophenol, PPh3, DIAD, dioxane, 78%; (ii) 7, DMF, NaI, 61%; (iii) 9, DMAP, DMF,
65%; (iv) (a) 11, HOAc, EtOH, 41%; (b) K2CO3, MeOH, H2O, 90%.
dA22ꢀdT22 with NHP was apparent. When compared with other
ligands, such as Hoechst 33258, NH, NP, and HPA, the triple-
binding agent NHP was clearly the strongest stabilizer of this
sequence polymers.19 DNA intercalation involves both unstacking
of base pairs to accommodate the intercalants and unwinding of
the double helix. The energy requirement for creating the interca-
lation sites is lower for alternating pyrimidine (30–50)-purine than
for a nonalternating homocopolymer used in this study. This ener-
getic cost for unwinding and unstacking is likely paid by the con-
current binding of neomycin and Hoechst 33258 in the two
grooves.
DNA oligomer, with a
DTm = 35 °C. A concentration–dependent
melting study (Fig. 2) illustrates the gradual shift in Tm values
as ligand concentrations are increased. These results indicate a
clear effect of each recognition element of NHP on DNA stability,
as solutions containing
a combination of individual ligands
(NH + aminoypyrene; neomycin + HPA; NP + Hoechst 33258)
exhibited significantly lower Tm values.
Circular dichroism (CD) was also utilized to probe the changes
in ligand and DNA conformation attributable to complexation.
Several CD studies have indicated the tight and rigid conforma-
tional preference of Hoechst 33258 when bound to A/T rich se-
quences, typically characterized by an increase in CD signal in
the region of Hoechst 33258 kmax. The current study supports that
observed previously by us and others4,11: a pronounced induced
(+) CD band around 360 nm as ligand concentrations increased,
consistent with previous CD reports of minor groove binding of
Hoechst 33258 and as also observed previously with Hoechst
33258-neomycin conjugates4,11 (Fig. 3). A noticeable overlap with
When these studies were carried out in the presence of a DNA
(dT) third strand, no triplex formation was observed with NH or
NHP, due to neomycin binding in the major groove, consistent with
previous reports.4,7,11 Of importance also is the fact that while ami-
nopyrene, NP, or HPA show no contribution of pyrene to an in-
crease in duplex Tm, its conjugation to neomycin and Hoechst
33258 in NHP clearly leads to a increase of about 11 °C in Tm over
NH. It is well known that pyrene favors the alternating purine–
pyrimidine sequence over the non-alternating purine–pyrimidine

B. Willis, D. P. Arya / Bioorg. Med. Chem. Lett. 19 (2009) 4974–4979
4977
N
NHR
N
O
HO
HO
HN
H N
RHN
N
R
O
NHR
O
+
S
OH
SCN
OH
O
NHR
HN
N
R = Boc
O
OH
O
RHN
HO
O
O
12
O
N
O
O
O
NH2
3
i) pyridine, DMAP
ii) TFA/CH2Cl2
N
N
HN
N
HN
N
NHR
O
HO
HO
RHN
O
N
H
O
O
S
O
NHR
R
O
N
O
S
OH
O
O
N
H
N
H
OH
O
NHR
13 R = Boc
"NHP"
O
OH
O
4
R = H
RHN
HO
Scheme 2. Preparation of triple recognition agent NHP. Reagents and conditions: (i) pyridine, DMAP, 52%; (ii) 1:1 TFA/CH2Cl2, quantitative.
a new induced CD signal is apparent between 270 and 400 nm,
*
Viscometry has long been utilized for investigating ligand–
substrate binding modes, particularly to confirm intercalation
events. Viscometric titrations of neomycin, Hoechst 33258, NH,
and NHP with poly(dA)ꢀpoly(dT) were thus carried out (Fig. 4).
In all cases except Hoechst 33258, the DNA solution viscosity de-
creased when ligand was added. The most marked decrease was
with NH, whereas NHP was clearly higher. Similar to that ob-
served before11,20 the decrease in viscosity can be attributed to
groove binding, which, in contrast to intercalation, can shorten
the helix by compaction. This compacting of DNA is clearly a re-
sult of neomycin interaction. Difference plots of NH–DNA binding
with NHP–DNA binding in the viscosity experiments were gener-
ated to study the effect of the pyrene moiety in NHP. By overlay-
indicative of an induced CD band likely due to a
p to p absorp-
tion of the pyrene moeity.9 Since asymmetry is induced to both
chromophores of NHP upon binding to DNA, these data support
the hypothesis that both the Hoechst 33258 and pyrene moieties
are interacting with DNA.
Importantly, it is known that pyrene binding to homocopoly-
mers such as poly(dA)ꢀpoly(dT) also occurs via an ‘external’, non-
intercalative binding mode that is characterized by little change
in Tm, viscosity, or circular dichroism in pyrene’s absorption re-
gion.21,19 Our observations with NHP in the presence of poly(-
dA)ꢀpoly(dT) suggest an intercalation mode similar to that
9,21
observed with intercalation events in poly(dA-dT)2.
Therefore,
it is likely that a combination of conformational restrictions in
NHP and the resulting unwinding of the duplex by concerted
groove binding of neomycin and Hoechst 33258 moieties designate
pyrene to an intercalative mode of binding. Almost no change is
seen in the CD spectrum between 200 and 260 nm, suggesting that
the DNA conformation is relatively unperturbed upon ligand
binding.
ing the subtracted data (NHP minus NH, Fig. 5), a clear
resemblance to theoretical intercalation is observed for the pyr-
ene moiety in NHP. The slightly lower slope can be attributed
to intercalation occurring less periodically within the DNA lattice
due to the large binding site size (one pyrene per every nine base
pairs) of NHP.11 Furthermore, the mere observation of viscosity
decrease, as seen in previous accounts,11 supports neomycin bind-

4978
B. Willis, D. P. Arya / Bioorg. Med. Chem. Lett. 19 (2009) 4974–4979
0.9
0.85
0.8
Tm2
48
48
70
ΔTm2
0
0
22
Ligand
None
Neomycin
Hoechst
33258
aminopyrene
NP
→
→
1
1
62
48
80
83
0.75
0.7
50
53
72*
64*
83
2
5
24*
16*
35
0.65
0.6
NH
HPA
NHP
0.55
20
30
40
50
60
70
80
90
NH +
aminopyrene
HPA +
neomycin
NP + Hoechst
33258
73
25
T(oC)
61*
69
13*
21
Figure 2. UV melting profiles of dA22ꢀdT22 with NHP. Samples of DNA (1
lM in duplex) were mixed with ligand (0–3 lM from left to right) in buffer (10 mM sodium
cacodylate, 0.5 mM EDTA, 150 mM KCl, pH 6.8) before heating at 95 °C for 5 min and slow annealing to 20 °C before UV analysis at 260 nm from 20 to 95 °C at heating rate of
*
0.2°/min. Tm reported is the midpoint temperature of dA22ꢀdT22 denaturation (right) Tm data for dA22ꢀdT22 melting in the presence of indicated ligands (3
l
M). For
compounds NH and HPA, biphasic transitions were observed (at unbound Tm of 48 °C and indicated Tm). See Supplementary data for solution conditions and UV melting
profiles.
15
1.2
10
inc. NHP
10
1.1
1
theoretical intercalation
Ht33258
5
0
inc. NH
5
0
0.9
0.8
0.7
-5
-5
NHP
NH
-10
-10
240
280
320
360
400
240
280
320
360
400
λ
λ
15
0
0.01 0.02 0.03 0.04 0.05 0.06 0.07
rdb
10
5
Figure 4. Viscometric analysis of poly(dA)ꢀpoly(dT) with various ligands: DNA
solutions (100 M) were titrated with respective drug (200 M) and corresponding
flow times were recorded in triplicate with deviation less than 0.1 s. Error bars are
indicated for each titration point. Buffer: 10 mM sodium cacodylate, 0.5 mM EDTA,
150 mM KCl, 1 mM MgCl2, pH 7.2; T = 27 0.05 °C.
l
l
0
1.15
-5
-10
NHP - NH (subtracted)
native
NH
NHP
1.1
240
280
320
360
400
intercalation
1.05
λ
Figure 3. Circular dichroism of poly(dA)ꢀpoly(dT) titrated with NH and NHP (top)
and an overlay of NH and NHP scans (below). A solution of DNA (30 M) was
titrated with small aliquots of concentrated ligand (200 M) before equilibration
and scanning from 450–210 nm. Peaks around 360 nm correspond to ligand–DNA
complexation. Solutions were in 10 mM sodium cacodylate, 0.5 mM EDTA, 150 mM
KCl, pH 7.2; T = ambient.
1
l
l
Theoretical Intercalation
subtracted
0.95
Hoechst 33258
0.9
0
0.01
0.02
0.03
0.04
0.05
rdb
ing. These data, from a combination of UV, flourescence, CD and
viscosity measurements, give strong candidacy for a DNA triple-
recognition agent in NHP.11 These studies should open the door
for development of novel DNA binding agents with specificities
and affinities far surpassing the current arsenal of DNA binding
drugs.
Figure 5. Subtracted viscosity data demonstrate pyrene effect on solution viscosity:
DNA solutions (100 M) were titrated with respective drug (200 M) and corre-
l
l
sponding flow times were recorded in triplicate with deviation less than 0.1 s.
Buffer: 10 mM sodium cacodylate, 0.5 mM EDTA, 150 mM KCl, 1 mM MgCl2, pH 7.2;
T = 27 0.05 °C.

B. Willis, D. P. Arya / Bioorg. Med. Chem. Lett. 19 (2009) 4974–4979
6. Charles, I.; Xi, H.; Arya, D. P. Bioconjugate Chem. 2007, 18, 160.
4979
Acknowledgement
7. Willis, B.; Arya, D. P. Adv. Carbohydr. Chem. Biochem. 2006, 60, 251.
8. Arya, D. P. Top. Curr. Chem. 2005, 253, 149.
The author is grateful for financial support from NSF (CHE/MCB-
0134972).
9. Hartley, J. A.; Webber, J.; Wyatt, M. D.; Bordenick, N.; Lee, M. Bioorg. Med. Chem.
1995, 3, 623.
10. Waring, M. J.; Bailly, C. J. Mol. Recognit. 1994, 7, 109.
11. Willis, B.; Arya, D. P. Biochemistry 2006, 45, 10217.
12. Arya, D. P.; Coffee, R. L., Jr.; Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001,
123, 5385.
Supplementary data
13. Xue, L.; Charles, I.; Arya, D. P. Chem. Commun. 2002, 70.
14. Arya, D. P.; Xue, L.; Tennant, P. J. Am. Chem. Soc. 2003, 125, 8070.
15. Shaw, N. N.; Arya, D. P. Biochimie 2008, 90, 1026.
16. Shaw, N. N.; Xi, H.; Arya, D. P. Bioorg. Med. Chem. Lett. 2008, 18,
4142.
17. Charles, I.; Arya, D. P. J. Carbohydr. Chem. 2005, 24, 145.
18. Charles, I.; Xue, L.; Arya, D. P. Bioorg. Med. Chem. Lett. 2002, 12, 1259.
19. Geacintov, N. E.; Shahbaz, M.; Ibanez, V.; Moussaoui, K.; Harvey, R. G.
Biochemistry 1988, 27, 8380.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.079.
References and notes
1. Bailly, C.; Henichart, J. P. Bioconjugate Chem. 1991, 2, 379.
2. Willis, B.; Arya, D. P. Curr. Org. Chem. 2006, 10, 663.
3. Hansen, M.; Hurley, L. J. Am. Chem. Soc. 1995, 117, 2421.
4. Arya, D. P.; Willis, B. J. Am. Chem. Soc. 2003, 125, 12398.
5. Arya, D. P.; Xue, L.; Willis, B. J. Am. Chem. Soc. 2003, 125, 10148.
20. Haq, I.; Ladbury, J. E.; Chowdhry, B. Z.; Jenkins, T. C.; Chaires, J. B. J. Mol. Biol.
1997, 271, 244.
21. Chen, F. M. Nucleic Acids Res. 1983, 11, 7231.