Programmable oligomers targeting 5′-GGGG-3′ in the minor groove of DNA and NF-κB binding inhibition

Article abstract of DOI:10.1016/j.bmc.2006.10.051

A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened to target 5′-WGGGGW-3′, a core sequence in the DNA-binding site of NF-κB, a prolific transcription factor important in biology and disease. Five Bi and Ip containing oligomers bound to the 5′-WGGGGW-3′ site with high affinity. One of the oligomers (Im-Im-Im-Im-γ-Py-Bi-Py-Bi-β-Dp) was able to inhibit DNA binding by the transcription factor NF-κB.

Full text of DOI:10.1016/j.bmc.2006.10.051

Bioorganic & Medicinal Chemistry 15 (2007) 759–770
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0
Programmable oligomers targeting 5 -GGGG-3 in the minor
groove of DNA and NF-jB binding inhibition
David M. Chenoweth, Julie A. Poposki, Michael A. Marques and Peter B. Dervan*
The Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
Received 13 September 2006; revised 23 October 2006; accepted 23 October 2006
Available online 26 October 2006
Abstract—A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened
0
0
to target 5 -WGGGGW-3 , a core sequence in the DNA-binding site of NF-jB, a prolific transcription factor important in biology
and disease. Five Bi and Ip containing oligomers bound to the 5 -WGGGGW-3 site with high affinity. One of the oligomers (Im-
0
0
Im-Im-Im-c-Py-Bi-Py-Bi-b-Dp) was able to inhibit DNA binding by the transcription factor NF-jB.
ꢀ
2006 Elsevier Ltd. All rights reserved.
5
1. Introduction
ring to lie over T not A. Since the development of these
pairing rules based on the five-membered heterocyclic
carboxamides Py, Im, and Hp, hundreds of DNA
sequences have been successfully coded for using polya-
DNA-binding polyamides based on the architecture of
the natural products netropsin and distamycin A have
emerged as a promising class of gene modulators.
1
,2
3,5
mides. Yet, due to sequence dependent alterations in
These molecules are capable of distinguishing the four
Watson-Crick base pairs in the DNA minor groove
and have been the subject of intense study along
with other classes of minor groove binders for interfer-
the shape of the DNA minor groove, there remain
DNA sequences that prove difficult to target with high
affinity and specificity.
3
c,d–f,4,5
ing with specific protein–DNA interfaces.
There has been an ongoing effort to broaden the reper-
toire of heterocycles with improved DNA recognition,
stability, and cellular trafficking profiles. We recently
reported that the benzimidazole ring can be an effective
platform for modular paired recognition elements in the
Sequence-specific recognition of the minor groove of
DNA by polyamides arises from the pairing of three dif-
ferent aromatic amino acids, pyrrole (Py), imidazole
4
,5
(
Im), and hydroxypyrrole (Hp). The direct readout,
8
,9
or information face, on the inside of the crescent-shaped
polyamide may be programmed by the incremental
change of atoms on the corners of the ring pairs present-
ed to the DNA minor groove floor. Stabilizing and
importantly, destabilizing interactions with the different
edges of the four Watson-Crick bases are modulated
by shape complementarity and specific hydrogen
minor groove of DNA. The benzimidazole 6–5 bicy-
clic ring structure, while having slightly different
curvature from the classic five-membered N-methyl pyr-
role–carboxamides, presents an ‘inside edge’ with a sim-
ilar atomic readout to the DNA minor groove floor,
8
,9
effectively mimicking Py, Im, and Hp. The imidazo-
pyridine/pyrrole pair Ip/Py distinguishes GÆC from
CÆG and the hydroxybenzimidazole/pyrrole pair Hz/Py
distinguishes TÆA from AÆT, providing a solution to
the unanticipated hydroxypyrrole instability limita-
4
–6,7l
bonds.
For example, the Im/Py pair distinguishes
GÆC from CÆG, TÆA, and AÆT. Im presents a lone pair
of electrons to the DNA minor groove and can accept
5
8,9
a hydrogen bond from the exocyclic amine of guanine.
Additionally, the Hp/Py pair distinguishes TÆA from
tion. This second generation solution to DNA recog-
nition can be elaborated further, deleting incrementally
almost all carboxamide linkages in the backbone of
the hairpin motif creating an oligomer comprised of four
4
–6
AÆT, GÆC, and CÆG. Hp projects an exocyclic OH
group toward the minor groove floor that is sterically
0
0
accommodated in the cleft of the TÆA base pair, prefer-
dimer units capable of binding the site 5 -GTAC-3 , a se-
quence formally containing all four Watson-Crick base
1
0
pairs. A key strategic issue for the program is interfer-
ing with protein–DNA interfaces in the promoter of
important genes. The new oligomer architecture has
*
Corresponding author. Tel.: +1 626 395 6002; fax: +1 626 683
753; e-mail: dervan@caltech.edu
8
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.051

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D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
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been successful in targeting the hypoxia inducible factor
HIF-1) binding site on the promoter of the VEGF
sequence 5 -WGGGGW-3 containing 4-consecutive
(
guanines (Fig. 3). Oligomers vary by incorporation of
benzimidazole–pyrrole into various positions in the par-
ent polyamide 1, resulting in oligomers 2–6. From previ-
ous studies, we expect the BiPy dimer to be a good
mimic for the traditional Py-Py recognition elements.
The Ip-Im moiety is introduced as a new mimic for
the traditional Im-Im recognition motif (Fig. 2). Quan-
1
1
gene. This recent success spurred our interest in using
these new oligomers to target another prolific transcrip-
1
2
tion factor, NF-jB, important in biology and disease.
Guanine rich sequences are highly prevalent and partial-
1
6
ly conserved in the promotor region of NF-jB driven
1
2a,13
genes (Fig. 1).
ry have established the ability of polyamides, targeting
Previous studies from our laborato-
1
7
titative DNase I footprinting titration experiments
0
0
the sequence 5 -GGGACT-3 , to inhibit DNA binding
by NF-jB, however targeting the four consecutive gua-
nines was less successful due to the modest affinity of
were used to determine the binding affinities and speci-
ficities against single base-pair mismatch sites of oligo-
mers 2–6 (Fig. 3) as compared to their parent
polyamide 1. We found that in oligomers 2–6, the 6–5
fused rings are effective mimics of their respective five-
membered ring systems and that these oligomers target
the binding-site 5 -WGGGGW-3 without loss of affini-
ty as compared to parent hairpin polyamide 1. In addi-
tion, we report the inhibition of DNA binding by the
transcription factor NF-jB using this new class of minor
groove binding oligomers (Figs. 3 and 4).
1
4,15
pyrrole/imidazole polyamides for this sequence.
The question arises whether a second generation oligo-
mer architecture can target 5 -GGGG-3 recognition
sequences with improved affinity.
0
0
0
0
We report the synthesis, DNA binding properties, and
NF-jB:DNA binding inhibition properties of a new
class of DNA binding oligomers targeted at the DNA
2. Results and discussion
2
.1. Heterocycle synthesis
Dimeric units Im-Ip-OH (9) and Boc-Im-Ip-OH (12)
were synthesized by oxidative condensation of aldehydes
7
and 10, respectively, with previously reported diami-
nopyridine 8. Mixing 7 and 8 in DMF at 80 ꢁC for 1 h
followed by 12 h of heating at 100 ꢁC in the presence
of FeCl and air afforded 9 after purification by precip-
3
itation from water and saponification using a mixture of
KOH (4 M) in MeOH at 40 ꢁC (Scheme 1). Nitroimi-
dazole (10) was synthesized from 7 using a mixture of
1
8
H SO + SO and neat red fuming nitric acid. 10 was
2
4
3
then added to a mixture of 8 in nitrobenzene and
refluxed at 140 ꢁC open to the atmosphere overnight
to provide NO -Im-Ip-OMe (11) after precipitation
from water. Compound 11 was reduced using Pd/C in
2
Figure 1. Crystal structure of the p50/p65 NF-jB heterodimer bound
0
0
to the DNA duplex 5 -TGGGGACTTTCC-3 (Ref. 13). The p50 and
p65 monomers are represented as gold and dark blue ribbons,
respectively. (a) Top view looking down the DNA double helix. (b)
Figure 2. Structures of the (a) pyrrole-benzimidazole internal dimer
(-Py-Bi-), (b) imidazole-imidazopyridine internal dimer (-Im-Ip-), and
(c) imidazole-imidazopyridine cap (Im-Ip-) in comparison with their
respective five-membered ring systems. Hydrogen-bonding surfaces to
the DNA minor-groove floor are bolded.
0
0
Side view showing the 5 -GGGG-3 oligomer binding site. GC rich
regions are shown in red and AT rich regions are shown in light blue.
(c) Sequence of DNA bound to NF-jB.

D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
761
Figure 3. Postulated hydrogen-bonding models for the 1:1 polyamide–DNA complexes with their matched sequence and the ball-and-stick
representation for compounds 1 and 6 over the 6-base-pair matched binding site (variable region W = A or T). (a) Im-Im-Im-Im-c-Py-Py-Py-Py-b-
Dp (1); (b) Im-Im-Im-Im-c-Py-Bi-Py-Bi-b-Dp (2), Im-Im-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (3), Im-Im-Ip-Im-c-Py-Bi-Py-Bi-b-Dp (4), Im-Ip-Im-Im-c-Py-
Bi-Py-Bi-b-Dp (5), Im-Ip-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (6).
Figure 4. Illustration of NF-jB:DNA binding inhibition by oligomer 2.
Scheme 1. Synthesis of imidazopyridine–imidazole dimers Im-Ip-OH
(
9) and Boc-Im-Ip-OH (12). Reagents and conditions: (i) 8, PhNO
40 ꢁC; (ii) oleum, red-fuming HNO ; (iii) 8, PhNO , 140 ꢁC; (iv) H
Pd/C, DMF; (Boc) O, DIEA, DMAP, DMF; 4 N KOH, MeOH.
2
,
,
the presence of hydrogen followed by Boc protection
using a mixture of Boc O and DMAP in DMF to pro-
1
3
2
2
2
2
vided the final dimer Boc-Im-Ip-OH (12) after saponifi-
cation and precipitation (Scheme 1).
40 ꢁC. Unreacted amines were acylated between cou-
2
.2. Oligomer synthesis
pling rounds using acetic anhydride. Deprotection of
the Boc-protected amines was accomplished using 80%
TFA in DCM. After completion of solid phase synthe-
sis, the resin was treated with dimethylaminopropyl-
amine (Dp) and the oligomers were purified by
reverse-phase preparatory HPLC: Im-Im-Im-Im-c-Py-
Py-Py-Py-b-Dp (1), Im-Im-Im-Im-c-Py-Bi-Py-Bi-b-Dp
(2), Im-Im-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (3), Im-Im-Ip-
Im-c-Py-Bi-Py-Bi-b-Dp (4), Im-Ip-Im-Im-c-Py-Bi-Py-
Oligomers 1–6 were synthesized using manual solid
phase synthesis methodology on commercially available
b-Ala-PAM resin as previously described (Scheme 2).
Starting from base resin (BR1), monomeric and dimeric
heterocyclic units were appended onto the resin in step-
wise fashion using HBTU activation. Couplings were al-
lowed to proceed for several hours between 25 and
1
9

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D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
Scheme 2. Representative solid-phase synthesis of polyamide 6 along with a table of the amino acid building blocks used for the synthesis. Reagents and
conditions:(i) 80% TFA/DCM; (ii) Boc-Py-Bi-OH(13), HBTU, DIEA, DMF; (iii) Ac O, DIEA, DMF; (iv) repeat (i–iii); (v) 80% TFA/DCM; (vi) Boc-c-
OH (14), HBTU, DIEA, DMF; (vii) Ac O, DIEA, DMF to provide BR1; (viii) 80% TFA/DCM; (ix) Boc-Im-Ip-OH (12), HBTU, DIEA, DMF; (x) 80%
TFA/DCM; (xi) Im-Ip-OH, HBTU, DIEA, DMF; (xii) dimethylaminopropylamine (Dp), 80 ꢁC 2 h; (xiii) preparative HPLC to give 6.
2
2
Bi-b-Dp (5), and Im-Ip-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (6).
Oligomers were characterized using MALDI-TOF mass
spectrometry.
and demonstrated minor selectivity (3-fold) over the
0
0
mismatch 5 -atGGGAt-3 , however showed an 11-fold
0
0
specificity over the mismatch 5 -atGAGGt-3 . Oligomer
demonstrated high affinity for the 4-G match sequenc-
4
9
ꢁ1
2
.3. DNA affinity and sequence specificity
es (K ꢀ mid 10 M ) with reasonable 5-fold selectivity
a
0
0
0
over the mismatch sequences (5 -AGGGAT-3 and 5 -
0
Quantitative DNaseI footprinting titrations were carried
out for oligomers 1–6 on the PCR product of plasmid
pEF16 (Figs. 5 and 6). Plasmid pEF16 was constructed
AGGAGT-3 ). Compounds 3 and 6 bound all designed
sequences with similar affinity. Thermodynamic data for
oligomers 1–6 are summarized in Table 1.
0
0
0
0
containing two designed match sites (5 -XGGGGT-3
X = A, T) and two mismatch sites (5 -AGGGAT-3
In general, the global increase in affinity for these novel
oligomers is not altogether unexpected. In contrast to
the 5-membered heterocyclic carboxamides, the 6–5
fused benzimidazole analogues have a larger hydropho-
bic surface, likely promoting their placement in the
0
0
and 5 -AGAGGT-3 (Figs. 5 and 6). The first two match
0
0
0
0
sites 5 -TGGGGT-3 and 5 -AGGGGT-3 were includ-
ed to determine if there was an energetic penalty associ-
0
0
ated with a 5 -AG-3 step. Previously, DNA sequences
0
0
21
containing multiple 5 -AG-3 transitions have proven
more difficult to target at high affinity and it has been
shown that changes in flanking sequence can affect bind-
DNA minor groove. Aromatic stacking and van der
2
Waals interactions are also contributing factors.
2,23
Furthermore, the benzimidazole derivatives are a more
rigid structure with a lower degree of rotational free-
dom. Such pre-organization may decrease the entropic
cost of DNA complexation.
2
0
0
ing at a proximal site. The second two binding sites, 5 -
0
0
0
AGGGAT-3 and 5 -AGAGGT-3 , were designed to
elucidate the energetic penalty for the loss of a favorable
hydrogen bond between the exocyclic amine of guanine
and the lone pair nitrogen on the oligomer in question.
2.4. NF-jB electrophoretic mobility gel shift assay
Control compound 1 bound both 4-G match sequences
0
The guanine rich region of the NF-jB binding element
has been shown to be important in protein–DNA recog-
0
(
5 -XGGGGT-3 , X = A, T) with comparably low affin-
8
ꢁ1
13,15,24,25
ity (K ꢀ 10 M ), showing no bias for either site
nition (Fig. 1).
Polyamides targeted to this re-
a
(Table 1). Compound 1 distinguished against mismatch
sequences (5 -AGGGAT-3 and 5 -AGAGGT-3 ) with
gion have previously been shown to inhibit NF-jB
binding. The possibility of steric or allosteric inhibi-
0
0
0
0
15
roughly 10-fold specificity (Table 1). Oligomer 2 demon-
strated a large increase in affinity (K ꢀ 10
tion exists due to numerous contacts between the p50
protein and the phosphate backbone or direct protein–
DNA contacts in the major groove, opposite the minor
groove polyamide binding site. To test whether the
second generation oligomer architecture would be
1
0
ꢁ1
M ) as
compared to 1 for both match sites, but showed lower
specificity (4-fold) over the mismatch sites. Oligomer 5
a
1
3,15
9
ꢁ1
)
showed a moderate increase in affinity (K ꢀ 10 M
a

D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
763
0
Figure 5. Quantitative DNase I footprinting experiments in the hairpin motif for polyamides 1, 2, and 3, respectively, on the 278 bp, 5 -end-labeled
PCR product of plasmid pEF16: lane 1, intact DNA; lanes 2 and 3, G and A sequencing reaction; lane 4, DNase I standard; lanes 5–15, 1 pM, 3 pM,
10 pM, 30 pM, 100 pM, 300 pM, 1 nM, 3 nM, 10 nM, 30 nM, and 100 nM polyamide concentration, respectively. Each footprinting gel is
accompanied by the following: (top) ball-and-stick models of the compound bound to its target DNA sequence; and (bottom) Binding isotherms for
the four designed sites. happ values were obtained according to published methods. Imidazoles and pyrroles are shown as filled and non-filled circles,
respectively; dimer units are represented as rectangles containing either Bi enclosed in a circle representing benzimidazole or Ip in a black box
representing imidazopyridine; beta alanine is shown as a diamond; the gamma-aminobutyric acid turn residue is shown as a semicircle connecting the
two subunits.
0
0
successful at targeting 5 -GGGG-3 in a biologically rel-
evant context we employed an NF-jB electrophoretic
mobility gel shift assay.
2 over a concentration range of 500 pM–500 nM estab-
lished an EC50 of 15.7 nM for NF-jB inhibition as
shown in Figure 8. In addition the identity of the NF-
jB band was confirmed by antibody supershift and the
data are shown in Figure 9.
The NF-jB binding inhibition properties of compounds
1
–6 were screened against a 40 bp DNA probe contain-
0
0
ing the 5 -WGGGGW-3 sequence, which is part of an
element from the intronic enhancer of the immunoglob-
ulin j light-chain gene recognized and bound by NF-jB
3. Conclusion
1
3
(
Fig. 7). In the initial screen each oligomer was tested
While traditional Py, Im, and Hp containing polyamides
have been successful at recognizing hundreds of pre-de-
termined DNA sequences with high affinity and specific-
at two concentrations, 10 and 100 nM, for the ability to
interfere with protein binding (Fig. 7). At the higher
concentration, 2 and 4 demonstrate a clear decrease in
band intensity, whereas compounds 1, 3, 5, and 6 have
little effect. This result agrees with the footprinting data,
as protein inhibition appears to scale with compound
affinity for the 5 -WGGGGW-3 site. At the 10 nM con-
centration compound 2 is the only compound to show
significant protein inhibition, reducing band intensity
by more than 80% (Fig. 7). Full titration of compound
0
0
ity, a host of target sequences such as 5 -GGGG-3 have
proven difficult to code for using polyamides. A series of
novel oligomers containing the 6–5 fused benzimidazole
(Bi) and imidazopyridine (Ip) heterocycles were devel-
oped. These oligomers, composed of 5-membered het-
erocyclic carboxamides, demonstrated a substantial
0
0
0
increase in affinity (10- to 100-fold) for the 5 -GGGG-
0
3 sequence. The marked increase in affinity could be

764
D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
0
Figure 6. Quantitative DNase I footprinting experiments in the hairpin motif for polyamides 4, 5, and 6, respectively, on the 278 bp, 5 -end-labeled
PCR product of plasmid pEF16: lane 1, intact DNA; lanes 2 and 3, G and A sequencing reaction; lane 4, DNase I standard; lanes 5–15, 1 pM, 3 pM,
10 pM, 30 pM, 100 pM, 300 pM, 1 nM, 3 nM, 10 nM, 30 nM, and 100 nM polyamide concentration, respectively. Each footprinting gel is
accompanied by the following: (top) ball-and-stick models of the compound bound to its target DNA sequnce; and (bottom) Binding isotherms for
the four designed sites. happ values were obtained according to published methods. Imidazoles and pyrroles are shown as filled and non-filled circles,
respectively; dimer units are represented as rectangles containing either Bi enclosed in a circle representing benzimidazole or Ip in a black box
representing imidazopyridine; beta alanine is shown as a diamond; the gamma-aminobutyric acid turn residue is shown as a semicircle connecting the
two subunits.
0
0
ꢁ1 a,b
(M )
Table 1. Affinities of 5 -GGGG-3 binding oligomers K
a
0
0
0
0
0
0
0
0
Polyamide
5 -atGGGGt-3
5 -aaGGGGt-3
5 -atGGGAt-3
5 -atGAGGt-3
8
8
7
7
1
1
2
4
2
1
.4(±1.0) · 10
.9(±1.4) · 10
.6(±0.6) · 10
.4(±0.9) · 10
.6(±0.5) · 10
.6(±0.2) · 10
2.6(±1.1) · 10
2.3(±0.8) · 10
61.0 · 10
1
9
9
9
9
0
10
9
9
9
2.0(±1.1) · 10
2.7(±0.4) · 10
4.8(±1.1) · 10
3.6(±0.9) · 10
9
9
2.6(±0.2) · 10
2.9(±0.1) · 10
9
8
8
4. 1(±1.2) · 10
8. 1(±2.3) · 10
8.2(±1.3) · 10
9
8
8
2.9(±0.2) · 10
8.6(±2.1) · 10
1.3(±0.1) · 10
2.5(±0.8) · 10
9
9
9
1.1(±0.4) · 10
1.1(±0.1) · 10
a
Values reported are mean values from at least three DNase I footprinting titration experiments, with the standard deviation given in parentheses.
Assays were performed at 22 ꢁC in a buffer of 10 mM Tris–HCl, 10 mM KC1, 10 mM MgCl, and 5 mM CaCl at pH 7.0.
b

D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
765
Figure 7. (a) Match DNA sequence with the p50 protein of NF-jB
overlapping the oligomer binding site. Asterisks indicate the location of
radio-labeled nucleotides in the probe sequence. (b) Mismatch DNA
sequence. (c) Gel shift screen for compounds 1–6 at concentrations of 10
and 100 nM. (d) Plot of relative NF-jB inhibition for compounds 1–6.
Figure 8. (Top) ball-and-stick model for 2 and EC50 value from gel
shift experiment. (Middle) representative NF-jB titration gel (n = 3)
for 2. (Bottom) binding isotherm for 2.
attributed to a combination of oligomer properties
including, a larger hydrophobic surface, a high degree
of ligand pre-organization, or differential solvation/des-
olvation effects. The ability of this class of new oligomers
to inhibit protein–DNA binding was demonstrated by
the inhibition of NF-jB. We are encouraged by the fact
that these oligomers demonstrate improved affinity for
guanine rich DNA sequences and future work directed
toward improving sequence specificity and examination
of the nuclear trafficking ability is a priority.
(Dp), Triethylamine (TEA), nitrobenzene (NO Ph), 2-
2
formyl-N-methylimidazole, red fuming nitric acid, 1,3-
dichloro-4-nitropyridine, 30% bromine in acetic acid, pal-
ladium acetate Pd(OAc) , and 10% palladium on carbon
2
were purchased from Aldrich. Boc-b-alanine-(4-carbonyl-
aminomethyl)-benzyl-ester-copoly(styrene–divinylben-
zene)resin (Boc-b-Pam-resin), dicyclohexylcarbodiimide
(DCC), hydroxybenzotriazole (HOBt), 2-(1H-ben-
zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate
(HBTU),
N,N-dimethylaminopyridine
(DMAP), and Boc-b-alanine were purchased from
NOVA Biochem. Trifluoroacetic acid (TFA) was pur-
chased from Halocarbon. All other solvents were reagent
grade from EM. Oligonucleotide inserts were synthesized
by the Biopolymer Synthesis Center at the California
Institute of Technology. Precoated silica gel plates
60F254 for TLC and silica gel 60 (40 lm) for flash chroma-
tography were from Merck. Glycogen (20 mg/ml), dNTPs
4
. Experimental
4
.1. General
N,N-Dimethylformamide (DMF), N,N-diisopropyleth-
ylamine (DIEA), N,N-dimethylaminopropylamine

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D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
4
.2. Heterocycle synthesis
Heterocyclic building blocks Boc-Im-OH and Boc-Py-
1
9,8
Bi-OH were synthesized as reported.
methyl-4-(1-methyl-1H-imidazole-2-carboxamido)-1H-
Im-Im-OH (1-
imidazole-2-carboxylic acid, CAS [464892-44-2]) and
Boc-Im-Im-OH
(4-(4-(tert-butoxycarbonylamino)-1-
methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-im-
idazole-2-carboxylic acid, CAS [502170-49-2]), are com-
mercially available.
4
.2.1.
1-Methyl-4-nitro-1H-imidazole-2-carbaldehyde
(
NO -Im-CHO) (10). A cooled flask (0 ꢁC) of 1-meth-
2
yl-2-imidazole-carboxaldehyde (7) (8 g, 72.6 mmol, Al-
drich) was treated dropwise with a precooled (0 ꢁC)
solution of red fuming nitric acid (75 ml) in conc.
H SO ÆSO (30%) (75 ml). The mixture was warmed to
2
4
3
room temperature and stirred for 12 h open to the atmo-
sphere. Next, the mixture was poured over ice, neutral-
ized with solid sodium carbonate, extracted four times
with dichloromethane, dried over anhydrous sodium
sulfate, and concentrated in vacuo to give a brownish-
yellow oil. The oil was recrystallized from i-PrOH/
Et O or EtOH/Et O to give 1-methyl-4-nitro-1H-imid-
2
2
azole-2-carbaldehyde (10) as a tan crystalline solid
(
1
4.5 g, 40% Yield). TLC (1:1 EtOAc/Hex) R = 0.4; H
f
NMR (300 MHz, DMSO-d ) d 9.74 (s, 1H), 8.71 (s,
6
1
3
1
H), 3.99 (s, 3H); C (75 MHz, DMSO-d ) d 182.31,
6
+
46.14, 140.56, 127.33, 35.70; HR-MS (EI ): Calcd for
1
C H O N : 155.0330. Found: 155.0350.
5
5
3
3
4
.2.2. 2-(1-Methyl-4-nitroimidazol-2-yl)-3H-imidazo[4,5-
b]pyridine-5-carboxylic acid methyl ester (NO -Im-Ip-
2
OMe) (11). 1-Methyl-4-nitro-1H-imidazole-2-carbalde-
hyde (2.01 g, 13.0 mmol) (10) and methyl 5,6- diamino-
Figure 9. Antibody supershift on match DNA. The NF-jB band is
shifted in the presence of anti-p50 or anti-p65 antibody.
pyridine-2-carboxylate
suspended in 120 ml of nitrobenzene were heated to
40 ꢁC for 48 h open to the atmosphere. The reaction
(8)
(2.17 g,
13.0 mmol)
1
(
PCR nucleotide mix), and all enzymes, unless otherwise
mixture was cooled to 23 ꢁC and the precipitate collect-
ed by vacuum filtration. The solid was washed with
diethyl ether and dried under high vacuum to provide
2-(1-methyl-4-nitroimidazol-2-yl)-3H-imidazo[4,5-b]pyr-
idine-5-carboxylic acid methyl ester (11) (3.6 g, 92%
stated, were purchased from Boehringer–Mannheim.
pUC19 was purchased from New England Biolabs, and
3
2
deoxyadenosine [c- P] triphosphate was provided by
ICN. Calf thymus DNA (sonicated, deproteinized) was
from Amersham Pharmacia. DNaseI (7500 U/ml, FPLC
pure) was purchased from Roche. AmpliTaq DNA poly-
merase was from Perkin-Elmer and used with the provid-
ed buffers. Tris–HCl, DTT, RNase-free water, and 0.5 M
EDTA were from United States Biochemical. Calcium
chloride, potassium chloride, and magnesium chloride
were purchased from Fluka. Tris–borate–EDTA was
from Gibco and bromophenol blue was from Acros. All
reagents were used without further purification. NMR
spectra were recorded on a Varian spectrometer at
1
yield) as a powdery tan solid. H NMR (300 MHz,
DMSO-d ) d 14.02 (broad s, 1H), 8.03–8.06 (m, 2H),
6
1
3
4.27 (s, 3H), 3.91 (s, 3H); C (75 MHz, DMSO-d ) d
6
166.01, 146.44, 142.95, 136.59, 135.80, 130.36, 127.35,
123.81, 121.17, 121.04, 52.85, 37.43; HR-MS (EI ):
+
Calcd for C H N O : 302.0760. Found: 302.0760.
1
2
10
6
4
4.2.3. 2-(4-Amino-1-methylimidazol-2-yl)-3H-imidazo[4,5-
b]pyridine-5-carboxylic acid methyl ester (H N-Im-Ip-
2
OMe).
2-(1-Methyl-4-nitroimidazol-2-yl)-3H-imidazo-
3
00 MHz in DMSO-d or CDCl with chemical shifts
6
[4,5-b]pyridine-5-carboxylic acid methyl ester (11) (3 g,
9.9 mmol) was dissolved in anhydrous DMF (150 ml)
and the solution was degassed with Ar. After the addition
of Pd/C (10 wt%, 600 mg) the reaction mixture was
purged three times with hydrogen and then left to stir at
23 ꢁC for 9 h under a hydrogen balloon atmosphere. After
filtering through a pad of Celite and washing with copious
amounts of EtOAc the filtrate was concentrated in vacuo
to give 2-(4-amino-1-methylimidazol-2-yl)-3H-imidazo-
[4,5-b]pyridine-5- carboxylic acid methyl ester, without
3
reported in parts per million relative to residual solvent.
UV spectra were measured on a Hewlett–Packard Model
8
FAB and EI mass spectra were recorded at the Mass Spec-
troscopy Laboratory at the California Institute of Tech-
nology. Matrix-assisted, laser desorption/ionization
time of flight mass spectrometry (MALDI-TOF-MS)
was conducted at the Mass Spectroscopy Laboratory at
the California Institute of Technology.
452 A diode array spectrophotometer. High resolution

D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
767
1
further purification (2.7 g, 100% yield). H NMR
(75 MHz, DMSO-d ) d 147.7, 142.0, 137.1, 131.8,
6
1
3
+
129.6, 126.9, 119.9, 109.3, 52.8, 35.8; HR-MS (EI ):
(
300 MHz, DMSO-d ) d; C (75 MHz, DMSO-d ) d
6
6
166.17, 149.03, 148.21, 141.40, 131.50, 120.12, 107.77;
HR-MS (EI ): Calcd for C H N O : 272.1020. Found:
Calcd for C H N O : 257.0910. Found: 257.0920.
1
2
11
5
2
+
1
2
12
6 2
2
72.1030.
4.2.7. 2-(1-Methylimidazol-2-yl)-3H-imidazo[4,5-b]pyri-
dine-5-carboxylic acid (Im-Ip-OH) (9). 2-(1-Meth-
ylimidazol-2-yl)-3H-imidazo[4,5-b]pyridine-5-carboxylic
acid methyl ester (Im-Ip-OMe) (250 mg, 0.97 mmol) dis-
solved in MeOH (2 ml) and KOH (4 N, 3 ml) was heated
to 50 ꢁC for 4 h. The methanol was removed in vacuo
and the aqueous layer washed with EtOAc (2· 10 ml)
to remove any starting material and trace impurities.
The pH of the aqueous layer was then adjusted slowly
to pH 4 with 1 N HCl upon which time a cloudy beige
precipitate formed. The mixture was placed in a falcon
tube and the precipitate concentrated by centrifugation.
The supernatant was decanted and the solid dried under
high vacuum to give 2-(1-methylimidazol-2-yl)-3H-imi-
dazo[4,5-b]pyridine-5-carboxylic acid (9) (154 mg, 65%
4
.2.4. 2-(4-[(tert-Butoxy)carbonylamino]-1-methylimid-
azol-2-yl)- 3H-imidazo[4,5-b]pyridine-5-carboxylic acid
methyl ester (Boc-Im-Ip-OMe). 2-(4-Amino-1-meth-
ylimidazol-2-yl)-3H-imidazo[4,5-b]pyridine-5- carboxyl-
ic acid methyl ester (11) (2.0 g, 7.3 mmol) dissolved in
DMF (25 ml) was treated with Boc O (5.3 g,
2
2
0
8
4.3 mmol), DIEA (5.2 ml), and DMAP (95 mg,
.73 mmol). The reaction mixture was then heated to
0 ꢁC for 72 h, cooled to 23 ꢁC, and flashed through a
plug of silica gel eluting with EtOAc to give a mixture
of mono- and di-bocked (2-(4-[(tert-butoxy)carbonyl-
amino]-1-methylimidazol-2-yl)-3-[(tert-butoxy)carbonyla-
mino]-imidazo[4,5-b]pyridine-5-carboxylic acid methyl
ester) products which were carried on for saponi-
fication.
1
yield) as a brown solid. H NMR (300 MHz, DMSO-
d ) d 7.99 (s, 2H), 7.51 (d, 1H, J = 0.9 Hz), 7.20 (d,
6
+
H, J = 0.9 Hz), 4.18 (s, 3H); HR-MS (EI ): Calcd for
1
4
.2.5. 2-(4-[(tert-Butoxy)carbonylamino]-1-methylimid-
C H N O : 243.0750. Found: 243.0740.
11 9 5 2
azol-2-yl)-3H-imidazo[4,5-b]pyridine-5-carboxylic (Boc-
Im-Ip-OH) (12). 2-(4-[(tert-Butoxy)carbonylamino]-1-
methylimidazol-2-yl)-3-[(tert-butoxy)carbonylamino]-imi-
dazo[4,5-b]pyridine-5-carboxylic acid methyl ester dis-
solved in MeOH (10 ml) and NaOH (1 N, 25 ml) was
heated to 50 ꢁC for 4 h. The reaction mixture was cooled
to 0 ꢁC and the pH adjusted slowly to pH 4 with 1 N
HCl. The reaction mixture was then extracted with ethyl
acetate (four times), dried over anhydrous sodium
sulfate, concentrated in vacuo, and dried under high
vacuum to give 2-(4-[(tert-butoxy)carbonylamino]-1-me-
thylimidazol-2-yl)-3H-imidazo[4,5-b]pyridine-5-carbox-
4.3. Oligomer synthesis
Oligomers were synthesized on solid support using Boc-
b-PAM resin (0.59 mequiv/g). Stepwise elongation of
the oligomers was done according to previously pub-
1
9
lished protocols. The synthesis of compound 1 has
4
been previously reported.
1
4.3.1. Preparation of base resin R-b-Bi-Py-Bi-Py-c-
NHBoc (BR1). To a manual solid phase synthesis vessel
was added Boc-b-PAM resin (0.3 g). The resin was
washed with DMF (15 ml) and allowed to swell for
15 min while shaking at room temperature. The resin
was then washed with DCM (ꢀ30 ml), followed by
80% TFA in DCM (ꢀ30 ml) to remove the Boc-group.
The resin was then agitated at room temperature in
80% TFA/DCM for another 25 min to provide the
1
ylic (12) (258 mg, 60% yield) as a light yellow solid. H
NMR (300 MHz, DMSO-d ) d 13.19 (br s, 2H), 9.53
6
(
(
s, 1H), 8.00 (m, 2H), 7.36 (s, 1H), 4.15 (s, 3H), 1.47
1
3
s, 9H); C (75 MHz, DMSO-d ) d 166.61, 152.92,
6
1
47.81, 142.48, 138.38, 132.01, 128.91, 119.74, 113.50,
9.07, 35.35, 28.12; HR-MS (EI ): Calcd for
+
7
C H N O : 358.1390. Found: 358.1370.
deprotected resin bound amine (R-b-NH ). Following
1
6
18
6
4
2
Boc-deprotection, the resin was washed with DCM and
10% DIEA in DMF to neutralize and prepare for cou-
pling. Simultaneously, in a separate reaction vessel,
Boc-Py-Bi-OH (189 mg, 531 lM), HBTU (191 mg,
504 lM), DIEA (137 mg, 185 ll, 1.06 mM), and DMF
(1.2 ml) were mixed and allowed to activate at room tem-
perature for 25 min. This mixture was then added to the
4
.2.6. 2-(1-Methylimidazol-2-yl)-3H-imidazo[4,5-b]pyri-
dine-5-carboxylic acid methyl ester (Im-Ip-OMe).
1
1
-Methylimidazole-2-carbaldehyde
(7)
.85 mmol) and methyl 5,6-diaminopyridine-2-carboxyl-
(214 mg,
ate (8) (310 mg, 1.85 mmol) were suspended in 17 ml
DMF and heated to 80 ꢁC for 1 h open to the atmo-
sphere. Next, FeCl –6H O (24 mg, 0.09 mmol) was add-
solid phase synthesis vessel containing R-b-NH . Cou-
3
2
2
ed and the reaction mixture was heated to 100 ꢁC for
2 h while air was bubbled through the reaction mixture.
pling was allowed to proceed at room temperature with
agitation for 3–6 h. Initial loading of the resin requires
elongated coupling times. Following coupling, the resin
was acylated by the addition of acetic anhydride to the
mixture and shaking for 15 min. The addition of the next
Boc-Py-Bi-OH (25) dimer was incorporated and depro-
tected as described above to provide the resin bound
1
The reaction mixture was then cooled to room tempera-
ture and poured over ice. The precipitate was collected
by filtration and washed with cold diethyl ether. The
material was dissolved in hot iso-propanol, cooled to
room temperature, and re-precipitated with diethyl
ether. The solid was washed with diethyl ether and dried
under high vacuum to provide 2-(1-methylimidazol-2-
yl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid methyl
fragment (R-b-Bi-Py-Bi-Py-NH ). To this fragment
2
was added a preactivated mixture of Boc-c-OH
(180 mg, 885 lM), HBTU (319 mg, 841 lM), and DIEA
(229 mg, 308 ll, 1.77 mM). Coupling was allowed to pro-
ceed for 3 h at room temperature with agitation. The res-
in was then capped with acetic anhydride as described
ester (Im-Ip-OMe) (240 mg, 50% yield) as s tan solid.
H NMR (300 MHz, DMSO-d ) d 7.99 (s, 2H), 7.51
1
6
1
3
(
s, 1H), 7.20 (s, 1H), 4.18 (s, 3H), 3.88 (s, 3H);
C

768
D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
above to provide the base resin R-b-Bi-Py-Bi-Py-c-
NHBoc (BR1). BR1 was then washed with DCM fol-
lowed by MeOH and Et O. The resin was then dried un-
2
reaction vessel and coupling was allowed to proceed
for 12 h at room temperature, followed by capping with
acetic anhydride as described for BR1 to give R-b-Bi-
Py-Bi-Py-c-Im-NHBoc. Following resin deprotection
as described above, Boc-Im-Ip-OH (12) was activated
as described for Boc-Im-OH. Coupling of 12 to the resin
was allowed to proceed overnight at room temperature
to provide R-b-Bi-Py-Bi-Py-c-Im-Ip-Im-NHBoc. Fol-
lowing resin deprotection as described above, Im-CCl3
(2-trichloroacetyl-1-methylpyrrole) (47 mg, 207 lmol)
and DIEA (53 mg, 72 ll, 413 lmol) were dissolved in
NMP (900 ll) and added to the reaction vessel. Cou-
der high vacuum and stored for subsequent use.
4
(
.3.2. Im-Im-Im-Im-c-Py-Bi-Py-Bi-b-Dp (2). BR1
50 mg) was added to a manual solid phase synthesis ves-
sel. The resin was washed with DCM (ꢀ15 ml), followed
by deprotection with 80% TFA in DCM. The resin was
shaken at room temperature in the 80% TFA solution
for 25 min. The resin was then drained, washed with
DCM, and neutralized with 10% DIEA in DMF. A
pre-activated mixture of Boc-Im-Im-OH (54 mg,
pling of Im-CCl to the resin was allowed to proceed
3
1
2
48 lM), HBTU (53 mg, 140 lM), DIEA (38 mg, 52 ll,
95 lM), and DMF (400 ll) was then added to the reac-
overnight at 32 ꢁC to provide R-b-Bi-Py-Bi-Py-c-Im-
Ip-Im-Im. The resin was treated with the cleavage proto-
col outlined below to provide Im-Im-Ip-Im-c-Py-Bi-Py-
Bi-b-Dp 4 in 3% Yield. MALDI-TOF-MS calcd for
tion vessel and coupling was allowed to proceed for three
hours at room temperature, followed by capping with
acetic anhydride as described for BR1 to give R-b-Bi-
Py-Bi-Py-c-Im-Im-NHBoc. Following resin deprotec-
tion as described above, Im-Im-OH was activated as de-
scribed for Boc-Im-Im-OH. Coupling of Im-Im-OH to
the resin was allowed to proceed overnight at room tem-
perature to provide R-b-Bi-Py-Bi-Py-c-Im-Im-Im-Im.
The resin was treated with the cleavage protocol outlined
below to provide Im-Im-Im-Im-c-Py-Bi-Py-Bi-b-Dp 2 in
+
C H N O : 1206.54. Found: 1206.50 [M+H] .
5
9
64 23
7
4.3.6. Im-Ip-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (6). BR1 (50 mg)
was added to a manual solid phase synthesis vessel. The
resin was treated with the cleavage protocol outlined be-
low to provide Im-Ip-Im-Ip-c-Py-Bi-Py-Bi-b-Dp 6 in 2%
yield. MALDI-TOF-MS Calcd for C H N O :
6
0
62 23
6
+
1200.52. Found: 1200.50 [M+H] .
5
1
% Yield. MALDI-TOF-MS: Calcd for C H N O :
58 66 23 8
+
212.55. Found: 1212.50 [M+H] .
4.4. Resin cleavage procedure
4
(
.3.3. Im-Ip-Im-Im-c-Py-Bi-Py-Bi-b-Dp (5). BR1
50 mg) was added to a manual solid phase synthesis
A sample of resin (20-100 mg) was washed with DCM
followed by the addition of dimethylaminopropylamine
(Dp) (1 ml). The mixture was heated to 80 ꢁC for 2 h
with occasional agitation. The resin was then filtered
and washed with 0.1% TFA in water (7 ml). The com-
bined filtrate was collected and subjected to purification
by reverse-phase preparatory HPLC using a Waters C18
column and 0.1% TFA/ACN solvent system. Appropri-
ate fractions from the HPLC purification were checked
for purity by analytical HPLC and characterized by
MALDI-TOF spectroscopy. Pure fractions were then
pooled, flash-frozen using liquid nitrogen, and lyophi-
lized to a dry solid for later use.
vessel and R-b-Bi-Py-Bi-Py-c-Im-Im-NHBoc was pre-
pared as described above for 2. Following deprotection,
washing, and neutralization as described above, a pre-
activated mixture of ImIp-OH (14) (21.5 mg, 88.5 lM),
HBTU (32 mg, 84 lM), DIEA (23 mg, 31 ll, 177 lM),
and DMF (400 ll) was added to the vessel containing
R-b-Bi-Py-Bi-Py-c-Im-Im-NH . Coupling was allowed
2
to proceed overnight at room temperature to provide
R-b-Bi-Py-Bi-Py-c-Im-Im-Ip-Im. The resin was treated
with the cleavage protocol outlined below to provide
Im-Ip-Im-Im-c-Py-Bi-Py-Bi-b-Dp 5 in 2.2% Yield.
MALDI-TOF-MS: Calcd for C H N O : 1206.54.
5
9
64 23
7
+
Found: 1206.60 [M+H] .
4.5. Footprinting experiments
4
(
.3.4. Im-Im-Im-Ip-c-Py-Bi-Py-Bi-b-Dp (3). BR1
50 mg, 0.81 mequiv/g) was added to a manual solid
Plasmid pEF16 was constructed using standard meth-
ods. DNaseI I footprint titrations were performed
according to standard protocols.
1
7
phase synthesis vessel. The resin was treated with the
cleavage protocol outlined below to provide Im-Im-
Im-Ip-c-Py-Bi-Py-Bi-b-Dp 3 in 3% Yield. MALDI-
TOF-MS: Calcd for C H N O : 1206.50. Found:
4.6. NF-jB electrophoretic mobility shift assay
5
9
64 23
7
+
1
206.50 [M+H] .
4.6.1. Materials. Jurkat nuclear extract containing acti-
vated NF-jB was purchased from Active Motif
(36013) and diluted as necessary just prior to use with
Buffer C (20 mM Hepes, pH 7.9, 420 mM KCl,
4
(
.3.5. Im-Im-Ip-Im-c-Py-Bi-Py-Bi-b-Dp (4). BR1
70 mg, 0.59 mequiv/g) was added to a manual solid
phase synthesis vessel. The resin was washed with
DCM (ꢀ15 ml), followed by deprotection with 80%
TFA in DCM. The resin was shaken at room tempera-
ture in the 80% TFA solution for 25 min. The resin
was then drained, washed with DCM, neutralized with
1.5 mM MgCl , 25% glycerol, 0.2 mM EDTA, 0.5 mM
2
DTT, 0.2 mM PMSF, and 1 lg/ml aprotinin). The final
working concentration was 0.5–2.5 lg/ll. The identity of
the NF-jB band was confirmed by antibody supershift
(Fig. 9) using antibodies against p50 (sc-7178, sc-114)
and p65 (sc-372) from Santa Cruz Biotechnology.
5
0% DIEA in DCM, and washed with DMF. A pre-ac-
tivated mixture of Boc-Im-OH (50 mg, 207 lmol),
HBTU (79 mg, 208 lmol), DIEA (53 mg, 72 ll,
4.6.2. Antibody supershift. The NF-jB antibody super-
shift data are shown in Figure 9. NF-jB antibodies
4
13 lmol), and DMF (900 ll) was then added to the

D. M. Chenoweth et al. / Bioorg. Med. Chem. 15 (2007) 759–770
769
(
sc-7178 and sc-114 against p50 and sc-372 against p65)
tures were loaded and resolved on non-denaturing 5%
acrylamide, 5% glycerol gels for 2–2.5 h at 150 V. The
gels were immediately dried and exposed to a phospho-
image storage plate for at least 8 h. The gel and running
buffer was 24.8 mM Tris Base, 190 mM glycine, and
1 mM 0.5 M EDTA. Gels were pre-run for 15–45 min
prior to loading.
came from Santa Cruz Biotechnology. The actin anti-
body was purchased from Sigma–Aldrich (A5441).
Antibodies were diluted to 0.1 lg/ll just prior to use
with ice-cold PBS. Complete binding reactions con-
tained 0.1 ng labeled probe, 10 mM Tris–HCl, pH 7.5,
5
0
0 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA,
.1 mg/ml poly(dI-dC), 1 ll antibody (0.1 lg), and 4 lg
(
2 ll) nuclear extract in a total volume of 10 ll. Nuclear
4.6.6. Gel shift titration. Reaction mixtures were pre-
pared as described above with a constant 0.1 ng probe
and 3 lg nuclear extract, except that 10· solutions of
polyamide were prepared to give a final concentration
range of 500 pM–500 nM.
extract was incubated with antibody at 4 ꢁC for 1 h fol-
lowed by addition of binding buffer and probe. The
complete reactions were allowed to incubate a further
3
0 min at room temperature, loaded onto pre-run 5%
acrylamide, 5% glycerol gels, and resolved for 2 h at
50 V. Gels were pre-run for 15–45 min prior to loading.
The gel and running buffer was 24.8 mM Tris Base,
1
4.6.7. Data analysis. A Typhoon 8600 Variable Mode
Imager was used to visualize the gels and band intensi-
ty was quantified using ImageQuant software version
1
90 mM glycine, and 1 mM 0.5 M EDTA.
5.1 from Molecular Dynamics. The fraction of DNA
4
.6.3. Sequence of gel shift probes. Oligonucleotides were
bound to NF-jB was calculated as the intensity ratio
of the NF-jB shifted band to the sum total of all
bands, shifted and unshifted. EC50 values were deter-
mined graphically as the polyamide concentration re-
quired to reduce NF-jB band intensity to half its
value. The data were plotted versus polyamide concen-
tration and fit to the Hill equation (n = 1) using Kale-
idagraph software.
purchased from Integrated DNA Technologies. The size
of both probes following labeling was 40 bp. The match
probe contained an imbedded jB site from the intronic
enhancer of the immunoglobulin j light-chain gene
(
underlined below). As a control, the sequence of the
mismatch probe contained a mutated jB that prevented
NF-jB binding (changes bolded below).
0
Match probe: 5 AATTCATGCAGTTGAGGGGACT
0
TTCCCAGGCATGCA 3
Match complementary sequence: 5 AGCTTGCATGCC
Acknowledgments
0
0
TGGGAAAGTCCCCTCAACTGCATG 3
0
We thank Eric J. Fechter for the preparation of plasmid
pEF16 and Thomas Leung for valuable discussions. We
thank The National Institutes of Health for grant sup-
port and a NRSA predoctoral training grant to J.A.P.
We also thank the Parsons Foundation for a fellowship
to M.A.M.
Mismatch Probe: 5 AATTCATGCAGTTGAGGTTA
0
CTTTCCCAGGCATGCA 3
Mismatch complementary sequence: 5 AGCTTGCATG
0
0
CCTGGGAAAGTAACCTCAACTGCATG 3
0
4
.6.4. Preparation of 3 labeled probes. In separate reac-
tions, 1.1 pmol of match or mismatch oligonucleotide
was annealed to an equal amount of its complementary
sequence by heating at 95 ꢁC for 1 min and cooling slow-
References and notes
0
ly to room temperature. The 3 ends were radiolabeled
1. (a) Olenyuk, B. Z.; Zhang, G.; Klco, J. M.; Nickols, N. G.;
Kaelin, W. G., Jr.; Dervan, P. B. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 16768; (b) Darnell, J. E. Nat. Rev.
Cancer 2002, 2, 740–748; (c) Pandolfi, P. P. Oncogene
2001, 20, 3116–3127; (d) Dervan, P. B.; Edelson, B. S.
Curr. Opin. Struct. Biol. 2003, 13, 284–299.
. (a) Finlay, A. C.; Hochstein, F. A.; Sobin, B. A.; Murphy,
F. X. J. Am. Chem. Soc. 1951, 73, 341–343; (b) Arcamone,
F. N. V.; Penco, S.; Orezzi, P.; Nicolella, V.; Pirelli, A.
Nature 1964, 203, 1064–1065.
. (a) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.;
Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
1376–1380; (b) Pelton, J. G.; Wemmer, D. E. Proc. Natl.
Acad. Sci. U.S.A. 1989, 86, 5723–5727; (c) Wemmer, D. E.
Annu. Rev. Biomol. Struct. 2000, 29, 439–461; (d) Buc-
hmueller, K. L.; Staples, A. M.; Howard, C. M.; Horick,
S. M.; Uthe, P. B.; Minh Le, N.; Cox, K. K.; Nguyen, B.;
Pacheco, K. A. O.; Wilson, D. W.; Lee, M. J. Am. Chem.
Soc. 2004, 127, 742–750; (e) Baraldi, P. G.; Bovero, A.;
Fruttarolo, F.; Preti, D.; Tabrizi, M. A.; Pavani, M. G.;
Romagnoli, R. Med. Res. Rev. 2004, 24, 475–528; (f)
Reddy, P. M.; Toporowski, J. W.; Kahane, A. L.; Bruice,
T. C. Bioorg. Med. Chem. Lett. 2005, 15, 5531–5536.
3
with P using Sequenase Version 2.0 (Amersham) and
2
3
2
a P dATP (Perkin-Elmer). The resulting labeled probes
were purified with G-50 Microspin Columns (Amer-
sham). Probe concentration was estimated at 0.8 ng/ll
assuming 100% recovery based on a calculated molecu-
lar weight of 24,662 g/mol. Just prior to use, labeled
probes were diluted in water to 0.1 ng/ll.
2
4
.6.5. Gelshift screen. Dry HPLC purified aliquots of
3
polyamide were dissolved in water and their concentra-
tion determined by measuring the absorbance at 310 nm
ꢁ
1
ꢁ1
(
e = 69,520 M
C
). 10· solutions (1 lM and 100 nM)
of each polyamide were prepared from serial dilutions of
the concentrated stock. Binding reactions contained
0
5
0
.1 ng labeled probe, 10 mM Tris–HCl, pH 7.5,
0 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA,
.1 mg/ml poly(dI-dC), 1 ll of 10· polyamide (final con-
centration 10 nM or 100 nM), and 3 lg (1–2 ll) nuclear
extract in a total volume of 10 ll. Reactions were al-
lowed to equilibrate at room temperature without nucle-
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