Synthesis and biophysical studies of hairpin polyamides targeting the Brn-3b and GATA-3 transcriptional sites

Article abstract of DOI:10.1515/HC.2010.002

Hairpin polyamide analogs of distamycin A ( 1 ) were designed and synthesized to evaluate their ability to bind 5 ' -ATAGA-3 ' and 5 ' -AGATA-3 ' sequences which are important elements for controlling the function of the Brn-3b and GATA-3 transcriptional factors, respectively. The hairpin polyamides are composed of pyrrole and imidazole units linked together via a γ -aminobutyrate (GABA) unit. Hairpins 2b (Py-Py-Im- γ -Py-Py-Py) and 2c (Im-Py-Py- γ -Py-Py-Py) were synthesized to target the respective Brn-3b and GATA-3 cognate sequences. Preliminary biophysical studies including thermal denaturation and circular dichroism were performed and the results ascertained the binding of hairpins 2a and 2b to their respective cognate DNA sequences.

Full text of DOI:10.1515/HC.2010.002

Heterocycl. Commun., Vol. 16(4-6), pp. 221–225, 2010 • Copyright © by Walter de Gruyter • Berlin • New York. DOI 10.1515/HC.2010.002
Synthesis and biophysical studies of hairpin polyamides
targeting the Brn-3b and GATA-3 transcriptional sites
nervous system where its inactivation was shown to produce
defects in central nervous system development (He et al.,
1989; Gerrero et al., 1993; Ninkina et al., 1993; Turner et al.,
1994). In addition, Brn-3b is also expressed in reproductive
tissues such as the cervix (Lillycrop et al., 1992; Ndisang
et al., 1998), testis (Budhram-Mahadeo et al., 2001), and
breast (Budhram-Mahadeo et al., 1998). Overexpression of
Brn-3b has been detected in human neuroblastomas (Smith
and Latchman, 1996; Latchman, 1997) and several human
breast tumors (Budhram-Mahadeo et al., 1999; Dennis et
al., 2001), suggesting a role for this transcription factor in
cancer. In several breast cancers, elevated levels of Brn-3b
have been determined to enhance cell proliferation and
tumor growth (Budhram-Mahadeo et al., 1999; Dennis et al.,
2001). A reduction in Brn-3b levels slows down such growth
effects (Budhram-Mahadeo et al., 1999; Dennis et al., 2001).
Additionally, elevated Brn-3b levels increase cancer cell
migration (Ishrad et al., 2004) and confer resistance to growth
arrest stimulus. For example, Brn-3b represses the BRCA1
tumor suppressor which normally induces cell cycle arrest
(Budhram-Mahadeo et al., 1999). In addition to these direct
effects on cell growth, Brn-3b can act indirectly by associat-
ing with other proteins (Budhram-Mahadeo et al., 1998). In
particular, Brn-3b associates with the estrogen receptor (ER)
and enhances the effect of this receptor at estrogen response
elements (Budhram-Mahadeo et al., 1998). For example,
the expression of HSP27, which can be directly activated by
either Brn-3b or ER, is maximal when both Brn-3b and ER
work cooperatively (Lee et al., 2005).
Balaji Babu, Hilary Mackay, Abby Prast, Kristin
Dittenhafer, Ryan Davis, Christopher Tronrud, Toni
Rice, Sameer Chavda and Moses Lee*
Department of Chemistry, Hope College, Holland, MI
49423, USA
*Corresponding author
e-mail: lee@hope.edu
Abstract
Hairpin polyamide analogs of distamycinA(1) were designed
and synthesized to evaluate their ability to bind 5′-ATAGA-3′
and 5′-AGATA-3′ sequences which are important elements
for controlling the function of the Brn-3b and GATA-3 tran-
scriptional factors, respectively. The hairpin polyamides are
composed of pyrrole and imidazole units linked together via a
γ-aminobutyrate (GABA) unit. Hairpins 2b (Py-Py-Im-γ-Py-
Py-Py) and 2c (Im-Py-Py-γ-Py-Py-Py) were synthesized to
target the respective Brn-3b and GATA-3 cognate sequences.
Preliminary biophysical studies including thermal denatur-
ation and circular dichroism were performed and the results
ascertained the binding of hairpins 2a and 2b to their respec-
tive cognate DNA sequences.
Keywords: Brn-3b; DNA; GATA-3; hairpin polyamides;
minor groove; transcriptional factors.
The transcriptional factor GATA-3 belongs to a family of
six transcriptional factors found in the human body. Each
of these GATA proteins has been linked to different types of
cancer, including pancreatic, breast, ovarian, gastric, and col-
orectal (Guo et al., 2005). GATA-3 expression is enhanced
in pancreatic cancer cells over non-cancerous cells (Gulbinas
et al., 2006). GATA-3 has also been studied as a prognostic
marker for breast cancer. This data shows that GATA-3 is a
transcriptional activator in breast tissue and has been shown
inside the nucleus of cancerous breast cells (Mehra et al.,
2005). However, there is still uncertainty on the exact role of
GATA-3 in the pathogenesis of cancer.
Owing to the connection of GATA-3 and Brn-3b in affect-
ing cancer growth, they represent excellent targets for the
design of novel gene-focused cancer control agents. The use
of polyamide analogs of distamycin 1 (Figure 1) to interrupt
the binding of transcriptional factors and to inhibit gene func-
tion is under intense investigation by the authors’ laboratory
and others (Mackay et al., 2008). Pyrrole (Py) and imida-
zole (Im) containing polyamides can be synthesized to bind
with high affinity and selectivity in the minor groove of the
Introduction
Recent advances in the chemical biology of the human ge-
nome have opened up new avenues for drug discovery by
medicinal chemists. Because biological characteristics such
as polymorphisms and gene expression profiles are closely
related to drug responses, tailor-made therapy might be re-
alized by knowledge-based design (Hurley, 2002). Many
challenges, such as genome-based drug development and
tailored cancer chemotherapy based on individual genomic
construction or ‘personalized medicine’ have been the
focus of considerable attention (Wittung-Stafshede, 1998).
Therefore, much effort has been directed at designing and
developing functional small molecules that selectively bind
DNA and recognize base-pair sequences, allowing for the
targeting of specific sequences (Denison and Kodadek,
1998).
The transcription factor Brn-3b is a member of the POU
family of transcription factors, and they share a common
bipartite DNA binding domain (Verrijzer and Van der Vliet,
1993). Brn-3b was originally identified to play a role in the
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222 B. Babu et al.
CH
N
3 O
CH
N
NH
O
3 O
Z
CH
N
3 O
N
H
H
H
N
Y
N
H
H2N
NH
N
H
N
O
X
O
H
O
N
CH3
NH2
H
N
H
N
N
H
O
HN
O
CH3
N
N
CH3
H
N
N
H3C
CH3
O
O
N
N
CH3
H3C
CH3
2a: X=CH; Y,Z=N;
2b: X,Y=CH; Z=N;
2c: X=N; Y,Z=CH;
1, distamycin A
Figure 1 Structures of distamycin 1 and hairpin polyamides 2a, 2b, 2c.
desired DNA sequence by following the established ‘pairing
rules’: Im/Py binds G⋅C and Py/Im binds C⋅G, Py/Py binds
either to A/T or T/A, and Im/Im binds T/G mismatched base
pairs (Kopka et al., 1997; Yang et al., 1999; Dervan and
Edelson, 2003; Lacy et al., 2004). Previous research by the
authors’ laboratory demonstrated that hairpin polyamide
2a (Figure 2) was capable of entering the cell nucleus and
inhibiting the action of nuclear factor Y (NF-Y) at the inverted
CCAAT box 2 (ICB2) site on the topoisomerase IIα (topo
IIα) promoter (Henry et al., 2004). Anticancer agents such
as etoposide and doxorubicin, which target topo IIα, become
ineffective in confluent cancer cells where the expression of
this enzyme is downregulated. NF-Y affects this downregula-
tion by binding to the ICB2 site (5′-ATTGGT-3′). As depicted
in Figure 2, hairpin 2a, which has a polyamide sequence Py-
Im-Im-γ-Py-Py-Py was designed to bind to the 3′-flank of this
sequence and it was found to interfere with NF-Y binding and
its downstream events. DNaseI footprinting studies showed
binding to the desired site and electrophoretic mobility shift
assay studies demonstrated inhibition of NF-Y binding to
ICB-2 (Henry et al., 2004). Incubation of confluent NIH 3T3
cells with 2a demonstrated increased expression of topo IIα
(Hochhauser et al., 2007). Following exposure to etoposide,
increased DNA double-stand breaks were observed by the
comet assay.
sequence 5′-AGATA-3′ (Figure 3). The binding characteris-
tics of 2b and 2c were investigated preliminarily using ther-
mal denaturation (∆Tm) and circular dichroism (CD) (Lacy et
al., 2002; Westrate et al., 2009).
Results and discussion
Synthesis of 2b and 2c
The synthetic approach towards obtaining polyamides 2b
and 2c is outlined in Scheme 1. The desired hairpin poly-
amides were synthesized using standard Schotten-Baumann
coupling of amines and acid chlorides. The bottom piece N-
(N′,N′-dimethylaminopropyl)-4-[4-(1-methyl-4-nitropyrole-2-
carboxamido)-1-methylpyrrole-2-carboxamido]-1-meth-
ylpyrrole-2-carboxamide 3 was synthesized using the previ-
ously described procedure in 95% yield (Matsumoto et al.,
1990).
Theintermediate11 wassynthesizedusingthereadilyavail-
able 1-methyl-4-nitro-2-trichloroacetylimidazole 4 (Jaramillo
et al., 2004). Reaction of 4 with ethyl γ-aminobutyrate
hydrochloride yielded the previously reported compound 5.
Reduction of 5 using 5% Pd/C in cold ethanol, followed by
coupling with acid chloride (prepared from 6 using SOCl2),
yielded compound 7, which in turn was reduced using the
same protocol as discussed above and coupled to acid 8 using
PYBOP, diisopropylethylamine (DIPEA) in dry DCM yield-
ing 10 in 25% yield. Hydrolysis of 10 followed by coupling
of the carboxylic acid 11 with the amine derived from hydro-
genation of 3 using PyBOP and DIPEA in dry DCM gave the
desired hairpin polyamide 2b.
Building on the success of the above strategy, the current
report describes the synthesis and evaluation of hairpin poly-
amides 2b and 2c (Figure 1), designed specifically to bind
the respective promoters and hopefully inhibit Brn-3b and
GATA-3. Altering the structure of 2a by replacing the second
Im group with Py (Py-Py-Im-γ-Py-Py-Py) produces hairpin 2b
that should recognize the sequence 5′-ATAGA located on the
3′-flank of the Brn-3b site (Figure 3). Replacing the top piece
with Im-Py-Py produces hairpin 2c that should recognize the
The intermediate 16 desired for the synthesis of 2c was
synthesized starting from 1-methyl-4-nitro-2-trichloroacetyl-
Figure 2 Target sequences for 2a, 2b and 2c.
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Hairpin polyamide analogs of distamycin A 223
Thermal denaturation (∆Tm) studies with hairpin 2c were
carried out using three DNA hairpins with a single binding
site representing the cognate, GATA site (5′-AGATA-3′), an
A/T rich non-cognate site (5′-AAATTT-3′), and a C/G rich
non-cognate site (5′-ACGCGT-3′). However, there was a
small change in thermal melt with the cognate sequence
(∆Tm=2°C), and interestingly it gave a higher melting temper-
ature with A/T rich non-cognate sequence (∆Tm=3°C). This is
not unusual especially considering that the cognate and A/T
rich non-cognate sequences differ by only one G/C base pair.
Imidazole-containing polyamides have been shown to toler-
ate and bind AT-rich sequences and the affinity is attributed
mainly to electrostatic attraction (Lown et al., 1986). AT-rich
sequences have the most negative molecular electrostatic
potential and the polyamides are cationic at physiological
pH (Lown et al., 1986).
Figure 3 Cognate and non-cognate DNA sequences for 2b and 2c.
pyrrole 12 (Xiao et al., 2000). Compounds 13 and 14 were
synthesized as described previously in the literature (Jaramillo
et al., 2004). Reduction of 14 using 5% Pd/C in cold etha-
nol, followed by coupling with acid chloride (prepared from
9 using SOCl2), yielded the unknown compound 15 in 95%
yield. Hydrolysis of 15 followed by coupling of acid 16 with
the amine derived from hydrogenation of 3 using PyBOP and
DIPEA in dry DCM gave the desired hairpin polyamide 2c.
Both the new hairpin polyamides 2b and 2c were character-
ized using IR, NMR and HRMS spectral data.
CD studies for compounds 2b and 2c
Thermal denaturation for compounds 2b and 2c
CD studies were carried out using the same DNA hairpins
described above. Minor groove binding of 2b to the cognate
sequence (5′-ATAGA-3′) (Figure 4A) is demonstrated by the
appearance of an induced CD band at 330 nm, which is typi-
cal of polyamides that bind in this manner (Lacy et al., 2002;
Hochhauser et al., 2007; Westrate et al., 2009). Interestingly,
binding was observed at the A/T rich non-cognate sequence
(Figure 4B). This is consistent with the ∆Tm studies as dis-
cussed above. It appears that 2b can tolerate this one base
mismatch, although binding to this sequence gave weaker
band than that of the cognate (Figure 4A), as suggested by
the reduced CD response at 330 nm (8 vs. 13 mdeg, respec-
tively). By comparison, no binding was observed to the C/G
rich non-cognate sequence (Figure 4C), demonstrating that
2b has some degree of selectivity.
Thermal denaturation (∆Tm) studies with hairpin 2b were
carried out using three DNA hairpins that contain a single
binding site representing the cognate, Brn-3b site (5′-
ATAGA-3′), an A/T rich non-cognate site (5′-AAATTT-3′),
and a C/G rich non-cognate site (5′-ACGCGT-3′). No change
in the melting temperature (∆Tm) between DNA alone and
DNA in the presence of compound 2b was observed for any
of the sequences. This was unexpected in the case of the
cognate site, as a melt had been observed with hairpin 2a
and the ICB2 sequence in previous studies (8°C) (Henry
et al., 2004). However, a similar result was observed with
other polyamides that have been shown to bind to its cog-
nate sequence by other biophysical methods but have shown
∆Tm values of 0°C. This is probably owing to low binding
affinity and the polyamide/DNA complex was not stable at
higher temperatures; hence, a ∆Tm was not observed.
CD studies were carried out using cognate DNA se-
quence for compound 2c. Minor groove binding of 2c to (5′-
(iii) (Y=CH)
Cl
(iii)
(ii)
Cl
O2N
CH3
OH
O
X
N
OH
O
Y
N
(ii)
NH2
CH3
O
O2N
O
O
NH2
Z
CH3
O2N
CH3
Y
O2N
H
H
N
Z
O
6
N
(8, X=N; 9, X=CH)
(i)
N
H3C
HN
O
CCl3
Z
N
CH3
N
O
(iv)
N
O
O
CH3
(iv) (X=CH)
(v) (X=N)
CH3
(4, Z=N; 12, Z=CH) (5, Z=N; 13, Z=CH)
(7, Y=CH; Z=N; 14, Y,Z=CH)
CH3
X
O
X
O
O
N
OH
N
H3C
(vi)
NH
H3C
(v)
NH
O
2b, X,Y=CH; Z=N
Y
H
N
O
HN
Y
H
2c, X=N; Y,Z=CH
X
HN
(ii)
Z
N
N
NO
N
H3C
O
CH3
H
N
N
O
O
CH3
H3C
N
O
NH2
N
H
N
O
CH3
CH3
N
N
H
N
O
(10, X,Y=CH; Z=N; 15, X=N; Y,Z=CH)
CH3
H3C
N
(11, X,Y=CH; Z=N; 16, X=N; Y,Z=CH)
O
3
CH3
Scheme 1 (i) Ethyl γ-aminobutyrate, dry Et3N, dry DCM, reflux, 18 h; (ii) 5% Pd/C, cold EtOH, rt; (iii) SOCl2, 15 min, reflux; (iv) dry Et3N,
dry DCM, 0°C-rt; (v) PyBOP, DIPEA, dry DCM, 2 days, rt; (vi) a: NaOH, H2O, MeOH, reflux, 30 min.
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224 B. Babu et al.
Figure 4 CD spectra of hairpin 2b with (A) the cognate sequence 5′-ATAGA-3′ and two non-cognate sequences, (B) 5′-A3T3-3′ and
(C) 5′-ACGCGT-3′. (D) CD spectra of hairpin 2c with cognate sequence 5′-AGATA-3′. Note that the x-axis represents wavelength (nm),
whereas the CD in mdeg is represented on the y-axis.
9.91 (s, 1H), 9.89 (s, 1H), 9.84 (s, 1H), 8.08 (br t, 1H), 8.04 (br t, 1H),
AGATA-3′) (Figure 4D) was demonstrated by the induced
7.50 (s, 1H), 7.33 (br d, 1H), 7.24 (d, 1H), 7.18 (d, J=1.2 Hz, 1H), 7.17
(d, J=2 Hz, 1H), 7.06 (d, J=1.6, 1H), 7.03 (d, J=1.6 Hz, 1H), 6.94 (m,
2H), 6.871 (d, J=1.6 Hz, 1H), 6.84 (s, 1H), 6.05 (br t, J=3.2 Hz, 1H),
3.93 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80
(s, 3H), 3.43 (quart, J=6 Hz, 2H), 3.19 (m, 2H), 2.54 (t, J=6.5 Hz, 2H),
2.28 (t, J=6 Hz, 2H), 2.21 (s, 6H), 1.81 (quin, 2H), 1.63 (quin, 2H);
ES+: 906 (M+); HRMS: Calcd., 906.4487; Obsd.: 906.4490.
CD band at 330 nm.
Conclusion
Small molecules that specifically bind to any sequence in the
human genome would be useful tools in molecular biology
and potentially in human medicine. Thus, two new hairpin
polyamides having some sequence specificity and binding
affinity towards their cognate DNA sequences, as indicated
by biophysical studies, have been synthesized and character-
ized. The goal in disclosing compounds 2b and 2c is to offer
the scientific community working in this area, an opportunity,
to utilize these compounds in biological studies.
Compound 2c
Yellow orange solid; yield=3%; mp: 230−237°C; IR (neat) 3733,
3584, 2921, 2850, 1715, 1556, 1540, 1506, 1471, 1456, 1435, 1404,
1
1260 cm-1; H NMR: (400 MHz, DMSO-d6) δ 10.46 (s, 1H); 10.29
(s, 1H); 9.93 (s, 1H); 9.92 (s, 1H); 9.89 (s, 1H); 9.85 (s, 1H); 8.55 (s,
1H); 8.32 (s, 1H); 8.07 (s, 1H); 8.07 (s, 1H); 7.59 (s, 1H); 7.39 (s,
1H); 7.29 (s, 1H); 7.24 (s, 1H); 7.18 (s, 1H); 7.16 (s, 1H); 7.04 (s,
1H); 7.03 (s, 1H); 6.91 (s, 1H); 6.88 (s, 1H); 6.82 (s, 1H); 3.99 (s,
3H); 3.84 (s, 6H); 3.83 (s, 3H); 3.80 (s, 3H); 3.79 (s, 3H); 3.24 (m,
2H), 3.19 (m, 2H); 2.33 (m, 2H); 2.23 (t, J=6.8 Hz, 2H), 2.13 (s, 6H);
1.79 (quin, J=6.4 Hz, 2H) 1.60, (quin, J=6.8 Hz, 2H); ES+: 906 (M+);
HRMS: Calcd., 906.4487; Obsd.: 906.4469.
Experimental
General procedure for the synthesis of 2b and 2c
Compound 3 (1 eq.) was reduced in the presence of hydrogen over
5% Pd/C (50% wt/wt) in cold MeOH (100 ml) at RT for ∼18 h. The
reaction mixture was filtered over celite and the catalyst was washed
thoroughly with MeOH. The solvent was removed by evaporation
and residual MeOH removed by co-evaporation with dry DCM
(3×2 ml). The resulting product was dried under high vacuum and
protected from light. Anhydrous 11 or 16 (1.2 eq.) and PyBOP (2.1
eq.) were added to the dry amine. The atmosphere was evacuated
out of the reaction mixture by purging with argon. This process is
repeated for two or three times to ensure the atmosphere is dry and
inert. The mixture was dissolved in dry DCM (∼10 ml) and diiso-
propylethylamine (3.1 eq.) was added. The reaction was allowed
to stir for 2 days in the dark with an argon balloon. A basic aque-
ous work-up was performed by using 2 m NaOH and the products
were extracted with DCM (3×20 ml). The combined organic layers
were washed with brine followed by water and dried over anhydrous
Na2SO4. The dried solution was evaporated under reduced pressure
to obtain the residue, which was purified by flash column chroma-
tography (silica, gradient 0:100−100:0 with ammonium hydroxide %
v/v, MeOH/CHCl3) to yield the desired compounds 2b or 2c as light
yellow orange/orange solids.
Acknowledgements
The authors are thankful to the support from the National Science
Foundation (CHE 0809162).
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