Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)?pyrrole and Hx?imidazole pairings

Article abstract of DOI:10.1016/j.bmcl.2013.01.075

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔT_M studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.

Full text of DOI:10.1016/j.bmcl.2013.01.075

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1699–1702
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hx-amides: DNA sequence recognition by the fluorescent Hx
(p-anisylbenzimidazole)ꢀpyrrole and Hxꢀimidazole pairings
Vijay Satam a, Pravin Patil a, Balaji Babu a, Matthew Gregory a, Michael Bowerman a, Mia Savagian a,
Megan Lee a, Samuel Tzou a, Kevin Olson a, Yang Liu b, Joseph Ramos b, W. David Wilson b,
John P. Bingham c, Kostantinos Kiakos c, John A. Hartley c, Moses Lee a,
⇑
a Division of Natural & Applied Sciences and Department of Chemistry, Hope College, Holland, MI 49423, USA
b Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
c Cancer Research UK Drug–DNA Interactions Research Group, UCL Cancer Institute, London WC1E 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst
33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies
of HxII (5) complete our studies on the first set of Hx-amides: Hx–I/P–I/P. HxPP (2), HxIP (3) and HxPI (4)
Received 2 December 2012
Revised 6 January 2013
Accepted 16 January 2013
Available online 25 January 2013
were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and
DTM studies showed that
Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to
mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly,
the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T–A/T; Hx/IP prefers
C–A/T; Hx/PI prefers A/T–C; and Hx/II prefers C–C. The results also showed that Hx-amides bound their
cognate sequence at a higher affinity than their formamido-triamide counterparts.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Hx-amides
DNA
Sequence recognition
Minor groove
Distamycin
Hoechst 33258
Polyamides are non-fluorescent imidazole (I) and pyrrole (P)-
containing analogs of distamycin A (1, Fig. 1). The small molecules
recognize and bind specific sequences of DNA within the minor
groove via a stacked, anti-parallel 2:1 ligand/DNA motif.1–3 A set
of pairing rules has been established for I and P-containing poly-
amide-DNA base pair recognition. A P/P pairing recognizes an A/T
or T/A base pair, an I/P pairing binds to a G/C base pair, while a
P/I pairing recognizes a C/G base pair. In addition, a stacked I/I pair-
ing targets a T/G mismatch but tolerates C/G or G/C.4–6 As a result,
polyamides can be tailored to bind any DNA sequence. These mol-
ecules have been applied to target the control regions of specific
genes, including those that cause cancer.7 A number of examples
have been reported,8 including reports from our laboratories on
NF-Y binding to ICB2 by these polyamides led to activation of the
repressed topo II gene in confluent cells. Ultimately, the confluent
a
cancer cells become sensitized to topo II
a targeted anticancer
drugs, such as etoposide.9,10 P- and I-containing polyamides are
therefore potentially useful for the development of treatments
for genetic or gene derived diseases, including cancer.
Despite the significant advancements that have been made in
the polyamide field, further improvements in the design and devel-
opment of novel polyamides are needed.11 Specifically, there is an
immediate need to develop polyamides that have good solubility in
water or aqueous biological media, readily localize in the nucleus,
and are fluorescent so they can be tracked in cells.11 A number of
fluorophores such as 40,6-diamidino-2-phenylindole (DAPI),12 thia-
zole orange,13 fluorescein,14 and bodipy15 have been incorporated
into the polyamide structure as a fluorescent tag. Although, such
fluorophore-polyamide hybrids have yielded interesting results,
this approach was compromised by two factors: first, the increase
in molecular mass of the conjugates and second, influence of fluo-
rescent tag on the cellular and nuclear uptake properties of the
conjugates.16 Ideally the polyamides should be inherently fluores-
cent yet maintaining an affinity and specificity for unique se-
quences. In 2007 Dervan’s group made some contributions to
this area by reporting hairpin polyamides that contained benz-
imidazole (Bz), imidazopyridine (Ip), and hydroxybenzimidazole
activation of the silenced topoisomerase IIa gene in confluent can-
cer cells.9,10 Specifically, we have demonstrated that low molecular
weight polyamides, such as f-PIP (Fig. 1)9 (f is the N-formamido
group found in distamycin) and PII-
were able to bind the Inverted CCAAT Box2 (or ICB2), which occurs
in the promoter of the topo II gene and is implicated in the con-
fluence-induced transcriptional downregulation of topoisomerase
c
-PPP,10 a hairpin polyamide,
a
IIa
through the binding of transcription factor NF-Y. Inhibition of
⇑
Corresponding author. Tel.: +1 616 395 7190; fax: +1 616 395 7923.
E-mail address: lee@hope.edu (M. Lee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.075

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V. Satam et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1699–1702
H3CO
CH3
N
NH2
O
N
CH3
HN
NH
N
NH2
H
HN
NH
HN
H
N
X
Y
H
N
O
O
O
HN
O
H3C
N
N
CH3
O
N
CH3
O
N
CH3
CH3
2, X = CH, Y = CH, n = 2, HxPP 3, X = N, Y = CH, n = 2, HxIP
1, Distamycin A
4, X = CH, Y = N, n = 1, HxPI
5, X = N, Y = N, n = 1, HxII
H3C
H3C
CH3
CH3
N
H
N
N
NH
O
H3C
NH
O
HN
HN
H
N
H
N
N
H
O
N
H3C
N
N
O
N
H3C
HN
O
N
O
N
CH3
N
O
N
CH3
CH3
O
CH3
f-PIP
PPIP
Figure 1. Structures of Distamycin A, 1, and the complete series of Hx–I/I–I/P:HxPP (2), HxIP (3), HxPI (4), HxII (5), along with f-PIP and PPIP.
Figure 2. Binding of Hx–I/P–I/P to their respective cognate sequences through the
stacked motif. (A) HxPP (2) and 50-AAATTT. (B) HxIP (3) and 50-ATCGAT. (C) HxPI (4)
and 50-ACATGT. (D) HxII (5) binding to its target sequence 50-ACCGGT.
CH3
H2N
N
N
N
CH3
H
N
HN
H3CO
N
OH
N
H
+
N
O
H3C
O
N
O
CH3
6
7
HOBt, EDCI.HCl,
TEA, DMF
rt, 16 h
5
Scheme 1. Synthesis of HxII (5).
(Hz) units. The emission property of these structural units allowed
for their detection by fluorescence studies.17
We have also been working on developing novel polyamides
that would overcome these challenges. In 2011, we reported the
design of a novel class of fluorescent and sequence specific binding
polyamides. These compounds contained a fluorogenic moiety,
p-anisylbenzimidazolecarboxamido. Hoechst 33258 inspired the
design of this fluorophore and we coined it ‘Hx’. Combining Hx
with polyamides created a new family of DNA binders, called
‘Hx-amides’. Three Hx-amides, HxPP (2), HxIP (3) and HxPI (4)
Figure 3. Autoradiogram of
Concentrations are expressed in
a
DNase
M.
I footprinting experiment of HxII (5).
l

V. Satam et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1699–1702
1701
presence of EDCI, hydroxybenzotriazole (HOBt), and triethylamine
at room temperature. Purification of the product by column chro-
matography using silica gel afforded the desired HxII as a yellow
solid. The compound was characterized by TLC, infrared,
400 MHz 1H NMR, and mass spectrometry.21
The sequence binding preference of HxII (5) was established
through a DNase I footprinting experiment using a 50-32P labeled
131 base pair DNA fragment that contain the cognate sequence
(50-ACCGGT). As shown in Figure 3, cleavage inhibition specifically
at the cognate site is observed at 10 lM and 30 lM. This result
demonstrates the selectivity of HxII for the target 50-ACCGGT se-
quence. The results provide direct evidence of the binding of HxII
to the target 50-ACCGGT site.
Binding of HxII to its preferred sequence 50-ACCGGT was further
corroborated by DNA thermal denaturation studies. Using 3 mol e-
quiv of the HxII-polyamide to DNA (bp),
DTM values of 5 °C, 0 °C,
and 5 °C were recorded for 50-AAATTT, 50-ACGCGT, and its cognate
sequence 50-ACCGGT, respectively. It is worth noting that a switch
of CG in the core site of ACCGGT to GC reduced the
DTM value to
0 °C. Binding of HxII to AAATTT is presumably due to electrostatic
stabilization of the positively charged polyamide and the polyan-
ionic backbone of the DNA. According to CD titrations studies
(Fig. 4), emergence of a strong positive DNA induced ligand band
at about 330 nm inferred the binding of HxII in the minor groove.22
The clear isodichroic point at about 310 nm for ACCGGT also sug-
gested that HxII bound through a single mechanism, presumably
in the minor groove and as a stacked dimer. In contrast, an isodich-
roic point was not apparent for AAATTT indicating the binding was
not by a single mechanism.
The biosensor-SPR method was employed to ascertain the bind-
ing affinity of HxII to its cognate and non-cognate sequences. The
studies were conducted using methodologies similar to those re-
ported for HxPP (2), HxIP (3), and HxPI (4).18 Thus the results are
directly comparable. The SPR results given in Figure 5 allowed
the determination of the binding constant of HxII for its cognate
50-ACCGGT to be 2 ꢁ 106 Mꢂ1. This result was calculated using a
model of 2:1 Hx-amides:DNA stoichiometry. The binding constants
for the non-cognate sequences are as follows: 50-ATGCAT,
1 ꢁ 106 Mꢂ1, and 50-AGCGCT, 5 ꢁ 105 Mꢂ1. Interestingly, these
results led to the following deductions: first, consistent with
Hx-amides 2–4, HxII (5) binds its cognate sequence with a higher
affinity than its non-cognate sequences. Second, its preference for
Figure 4. CD titration studies of HxII (5) to its cognate sequence 50-ACCGGT and to
a non-cognate sequence 50-AAATTT. CD experiments were carried out using 160
of a 9 M DNA solution, which was titrated with 1 mol equiv of 5, past the point of
saturation.
l
L
l
(Fig. 1) were recently reported.18 These compounds bound in the
minor groove and preferentially at specific sequences via the
stacked dimer motif. Specifically, HxPP preferred 50-AAATTT and
HxPI preferred 50-ACATGT. HxIP bound selectively to 50-ATCGAT
and 50-TACGAT.18 The latter sequence is located at the 50-flank of
the ICB2 site of the topoisomerase II
a promoter. Results from
Hx-amides 2–4 suggest that the Hx-moiety behaved comparably
to two consecutive pyrroles or PP in polyamides. In this communi-
cation, we complete the studies on the first set of Hx-amides Hx–P/
I–P/I by synthesizing the fourth molecule, HxII (5) (Fig. 1). Accord-
ing to the model in which Hx-amides bind their cognate sequences
given in Figure 2, HxII is expected to target 50-ACCGGT-30 DNA
sequence.
HxII polyamide (5) was synthesized according to the reaction
depicted in Scheme 1. 2-(4-Methoxyphenyl)benzimidazole-6-car-
boxylic acid (6)19 was coupled with amino-ImIm-N,N-dimethyl-
2-aminoethylamine (7)20 in anhydrous dimethylformamide in the
Figure 5. (A) SPR sensograms of HxII upon binding to its cognate sequence 50-ACCGGT. (B) Fitting of steady-state RU versus concentration using a two-site model. The steady-
state values at the lowest concentrations were determined by prediction from kinetics fits to the full curves.

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V. Satam et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1699–1702
50-ACCGGT as determined by SPR and other studies provided fur-
ther evidence that Hx behaves similarly to two consecutive pyr-
roles, PP. Third, Hx-amides produced enhanced binding affinity
over their formamido counterparts. For example, the binding con-
stants of HxIP (3), f-PIP, and PPIP (Fig. 1) for 50-ATCGAT were
2 ꢁ 106 Mꢂ1, 2 ꢁ 105 Mꢂ1, and 2 ꢁ 104 Mꢂ1, respectively.18 In this
study, HxII (5) also bound two-times more strongly to its cognate
M.; Chou, J. C.; Lefebvre, S.; Bando, T.; Shinohara, K.; Gottesfeld, J. M.; Sugiyama,
H. Bioorg. Med. Chem. 2010, 18, 168.
9. Le, M. N.; Sielaff, A.; Cooper, A. J.; Mackay, H.; Brown, T.; Kotecha, M.; O’Hare,
C.; Hochauser, D.; Lee, M.; Hartley, J. A. Bioorg. Med. Chem. Lett. 2006, 16, 6161.
10. (a) Henry, J. A.; Le, N. M.; Nguyen, B.; Howard, C. M.; Bailey, S. L.; Horick, S. M.;
Buchmueller, K. L.; Kotecha, M.; Hochhauser, D.; Hartley, J. A.; Wilson, W. D.;
Lee, M. Biochemistry 2004, 43, 12249; (b) Kotecha, M.; Kluza, J.; Wells, G.;
O’Hare, C. C.; Forni, C.; Mantovani, R.; Howard, P. W.; Morris, P.; Thurston, D. E.;
Hartley, J. A.; Hochhauser, D. Mol. Cancer Ther. 2008, 7, 1319.
11. (a) Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 12063; (b) Nishijima, S.; Shinohara, K.; Bando, T.; Minoshima,
M.; Kashiwazaki, G.; Sugiyama, H. Bioorg. Med. Chem. 2010, 18, 978; (c) Liu, B.;
Kodadek, T. J. Med. Chem. 2009, 52, 4604.
sequence
50-ACCGGT
than
f-PII:
2 ꢁ 106 Mꢂ1
versus
1 ꢁ 106 Mꢂ1 23
,
respectively. Fourth, given the inherent fluores-
cence property of Hx-polyamides, including HxII (excitation at
320 nm and emission at 360 nm), Hx-amides are readily traceable
in cells (data will be reported elsewhere). Fifth, as hydrochloride
salts Hx-amides readily dissolved in water at 1 mM. This is a signif-
icant improvement over the formamido triamides and the non-
formamido compounds.
12. Kapuschinski, J.; Yanagi, K. Nucleic Acids Res. 1979, 6, 3535.
13. Fechter, E. J.; Olenyuk, B.; Dervan, P. B. J. Am. Chem. Soc. 2005, 127, 16685.
14. Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 12063.
15. Belitsky, J. M.; Leslie, S. J.; Arora, P. S.; Beerman, T. A.; Dervan, P. B. Bioorg. Med.
Chem. 2002, 10, 3313.
16. (a) Edelson, B. S.; Best, T. P.; Olenyuk, O.; Nickols, N. G.; Doss, R. M.; Foister, S.;
Heckel, A.; Dervan, P. B. Nucleic Acids Res. 2004, 32, 2802; (b) Hsu, C. F.; Dervan,
P. B. Bioorg. Med. Chem. Lett. 2008, 18, 5851; (c) Nishijima, S.; Shinohara, K.;
Bando, T.; Minoshima, M.; Kashiwazaki, G.; Sugiyama, H. Bioorg. Med. Chem.
2010, 18, 978; (d) Crowley, K. S.; Phillion, D. P.; Woodard, S. S.; Schweitzer, B.
A.; Singh, M.; Shahany, H.; Burnette, B.; Hippenmeyer, P.; Heitmeier, M.;
Bashkin, J. K. Bioorg. Med. Chem. Lett. 2003, 13, 1565.
17. Chenoweth, D. M.; Viger, A.; Dervan, P. B. J. Am. Chem. Soc. 2007, 129, 2216.
18. Chavda, S.; Liu, Y.; Babu, B.; Davis, R.; Sielaff, A.; Ruprich, J.; Westrate, L.;
Tronrud, C.; Ferguson, A.; Franks, A.; Tzou, S.; Adkins, C.; Rice, T.; Mackay, H.;
Kluza, J.; Tahir, S. A.; Lin, S.; Kiakos, K.; Bruce, C. D.; Wilson, D. W.; Hartley, J. A.;
Lee, M. Biochemistry 2011, 50, 3127.
Finally, the results provide evidence that Hx functions as a PP
doublet and a set of pairing rules for DNA sequence recognition
is summarized in Figure 2. Specifically, Hx/PP pairing preferentially
binds in the minor groove of two A/T base pairs, or A/T–A/T
(Fig. 2A). The Hx/IP pairing binds a C–A/T doublet (Fig. 2B), Hx/PI
recognizes A/T–C, and Hx/II binds C–C.
In conclusion, we have completed the synthesis and DNA bind-
ing studies of all four members of Hx–I/P–I/P set of Hx-amides. The
results provide evidence that Hx is a versatile and useful DNA se-
quence recognition entity. Our group is actively engaged in opti-
mizing and further enhancing its sequence recognition
properties. Biological studies of Hx-amides are underway and the
results will be reported in due course.
19. Bathini, Y.; Lown, J. W. Synth. Commun. 1990, 20, 955.
20. Lee, M.; Rhodes, A.; Wyatt, M. D.; Forrow, S.; Hartley, J. A. Biochemistry 1993,
32, 4237.
21. p-Anisylbenzimidazole-imidazole-imidazole-dimethylaminoethylamine (HxII)
(5): 2-(4-methoxyphenyl)benzimidazole-6-carboxylic acid (6)18,19 (0.051 g,
0.19 mmol) was dissolved in anhydrous DMF (1.5 mL), and EDCIꢃHCl (0.11 g,
0.6 mmol) and dry triethylamine (0.11 mL, 0.8 mmol) were added under
nitrogen atmosphere at room temperature. The reaction mixture was stirred
for 5 min at room temperature and HOBt (0.087 g, 0.6 mmol) was added and
the mixture was stirred for another 5 min. A solution of amino-ImIm-N,N-
dimethyl-2-aminoethylamine (7)20 (0.053 g, 0.16 mmol) dissolved in
anhydrous DMF (2.0 mL) was added at room temperature. After the mixture
was stirred at room temperature for 16 h the reaction was complete, as
monitored by TLC [CH3OH:NH4OH:CHCl3 (1:0.15:5.5)], the solvent was
removed under vacuum (45–50 °C). The residue was purified by silica gel
column chromatography using a gradient CHCl3/MeOH (0:100–100:0%, v/v)
solvent system. The product 5 was isolated as a yellow solid (8.0 mg, 8.3%);
mp: 135–138 °C; Rf: 0.59 (1:0.15:5.5 v/v, CH3OH:NH4OH:CHCl3); FT-IR: 3256,
2960, 1630, 1572, 1527, 1467, 1431, 1259, 1152, 1098, 987, 880, 807, 740,
Acknowledgments
The authors thank the NSF (CHE 0809162, CHE 0922623), Can-
cer Research UK (C2259/A9994 to J.A.H.) and the Georgia Research
Alliance for their generous support.
References and notes
1. Dwyer, T. J.; Geierstanger, B. H.; Bathini, Y.; Lown, J. W.; Wemmer, D. E. J. Am.
Chem. Soc. 1992, 15, 5911.
2. Mrksich, M.; Wade, W. S.; Dwyer, T. W.; Geierstanger, B. H.; Wemmer, D. E.;
Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7586.
3. Pelton, J. G.; Wemmer, D. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5723.
4. (a) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215; (b) Kielkopf, C. L.; Baird, E. E.;
Dervan, P. B.; Rees, D. C. Nat. Struct. Biol. 1998, 5, 104.
5. Lown, J. W.; Krowicki, K.; Bhat, U. G.; Skorobaty, A.; Ward, B.; Dabrowiak, J. C.
Biochemistry 1986, 25, 7408.
6. Yang, X.-L.; Hubbard, R. B.; Lee, M.; Tao, Z.-F.; Sugiyama, A.; Wang, H.-J. Nucleic
Acids Res. 1999, 27, 4183.
7. Melander, C.; Burnett, J. M.; Gottesfield, J. J. Biotechnol. 2004, 112, 195.
8. (a) Chou, C. J.; Farkas, M. E.; Tsai, S. M.; Alvarez, D.; Dervan, P. B.; Gottesfeld, J.
M. Mol. Cancer Ther. 2008, 7, 769; (b) Tsai, S. M.; Farkas, M. E.; Chou, C. J.;
Gottesfeld, J. M.; Dervan, P. B. Nucleic Acids Res. 2007, 35, 307; (c) Minoshima,
696 cmꢂ1 1H NMR (CD3OD): d 8.20 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.83 (d,
;
J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.45 (s, 1H), 7.09 (d,
J = 8.0 Hz, 2H), 4.08 (s, 3H), 4.01 (s, 3H), 3.88 (s, 3H), 3.49 (t, J = 7.0 Hz, 2H), 2.56
(t, J = 7.0 Hz, 2H), 2.31 (s, 6H); LRMS (ES+) m/z 585 (M+H+, 100%); HRMS [M+H]+
calcd for m/z C29H32N10O4: 585.2686, obsd. 585.2706.
22. (a) Westrate, L.; Mackay, H.; Brown, T.; Nguyen, B.; Kluza, J.; Wilson, W. D.; Lee,
M.; Hartley, J. A. Biochemistry 2009, 48, 5679; (b) Lyng, R.; Rodger, A.; Norden,
B. Biopolymers 1992, 32, 1201.
23. (a) Lacy, E. R.; Minh Le, N.; Price, C. A.; Lee, M.; Wilson, W. D. J. Am. Chem. Soc.
2002, 124, 2153; (b) Buchmueller, K.; Staples, A. M.; Uthe, P. B.; Howard, C. M.;
Pacheco, K. A. O.; Cox, K. K.; Henry, J. A.; Bailey, S. L.; Horick, S. M.; Nguyen, B.;
Wilson, D. W.; Lee, M. J. Am. Chem. Soc. 2005, 127, 742.