Synthesis and duplex DNA recognition studies of oligonucleotides containing a ureido isoindolin-1-one homo-N-nucleoside. A comparison of host-guest and DNA recognition studies

Article abstract of DOI:10.1016/j.bmc.2003.12.022

In an effort to construct non-natural bases to be used in triplex-based antigene DNA recognition strategies, a uriedo-isoindolin-1-one homo-N-nucleoside base was designed to bind the cytosine-guanine (CG) base pair. An organic soluble analogue of this bas

Full text of DOI:10.1016/j.bmc.2003.12.022

Bioorganic & Medicinal Chemistry 12 (2004) 1517–1526
Synthesis and duplex DNA recognition studies of oligonucleotides
containing a ureido isoindolin-1-one homo-N-nucleoside. A
comparison of host–guest and DNA recognition studies
Eric Mertz, Sebastiano Mattei and Steven C. Zimmerman*
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Matthews Avenue, Urbana, IL 61801, USA
Received 5 August 2003; accepted 18 December 2003
Abstract—In an effort to construct non-natural bases to be used in triplex-based antigene DNA recognition strategies, a uriedo-
isoindolin-1-one homo-N-nucleoside base was designed to bind the cytosine-guanine (CG) base pair. An organic soluble analogue
of this base was shown to effectively complex CG (Kassoc=740 Mꢀ1) in chloroform through formation of three hydrogen bonds
(Mertz, E.; Mattei, S.; Zimmerman, S. C. Org. Lett. 2000, 2, 2931–2934). The novel nucleoside base was synthesized and success-
fully incorporated into oligonucleotides which were used in triple helix melting temperature studies. Low melting temperatures were
observed when the isoindolin-1-one base was paired opposite CG as well as GC, TA, and AT, thus indicating that despite favorable
recognition in model studies, the artificial base did not effectively recognize duplex DNA to form pyrimidine-purine-pyrimidine type
triple helices.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
ficial nucleoside bases for the selective recognition of the
CG and TA base pairs.6b,7,8 These non-natural bases
have ranged from modified natural bases to entirely
synthetic receptors. However, once incorporated into
oligonucleotide sequences, these novel bases have typi-
cally exhibited at best moderate affinity and selectivity
for their intended base pair targets.
Formation of DNA triple helices is a potentially pow-
erful approach to sequence-specific DNA binding.1
Indeed, binding of a certain single stranded oligonu-
cleotide (triplex-forming oligonucleotide or TFO)
within the major groove of the target duplex DNA can
achieve regulation of gene expression,2 site-directed
DNA modification,3 and controlled DNA cleavage.4,5
Despite these remarkable accomplishments, the wider
use of the triplex strategy is hampered by the fact that
none of the four natural bases recognize the pyrimidine
bases thymine (T) and cytosine (C) in the pyrimidine-
purine-pyrimidine (pyr-pur-pyr) motif.6 Thus, forma-
tion of the thymine-adenine-thymine (T-AT) and cyto-
sine-guanine-cytosine (C-GC) base triplets allows triplex
recognition to occur only over homopurine tracts of
duplex DNA. (Fig. 1; C denotes 20-deoxycytidine pro-
tonated at the 3-position nitrogen).
Three key considerations in the design of CG or TA
selective nucleosides are (1) optimization of hydrogen
bonding between the artificial base and the targeted
base pair, (2) maximization of p-stacking between the
artificial base and the adjacent base triplets, and 3) iso-
morphism between the non-natural base triplet and the
canonical T-AT and C-GC base triplets.1d,e In develop-
ing new artificial nucleoside bases for the recognition of
the CG base pair, we addressed the first two of these
criteria by designing aromatic, heterocyclic bases cap-
able of forming three hydrogen bonds with the major
groove (Hoogsteen) side of the CG base pair.9 The third
design criterion (isomorphism) was addressed by com-
paring two-dimensional and three-dimensional models
of proposed base triplets with the canonical T-AT and
C-GC triplets. In this analysis, the position of the 20-
deoxyribose groupof the artificial base should match
closely with the position of the 20-deoxyribose groupin
the TFO strand of T-AT or C-GC.
To expand the scope of triple helix technology, intense
efforts have focused on the design and synthesis of arti-
Keywords: DNA Recognition; Nucleoside; Triplex DNA.
* Corresponding author. Tel.: +1-121-733-36655; fax: +1-121-724-
49919; e-mail: scz@scs.uiuc.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.022

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E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
We have investigated the heterocyclic hydrogen bonding
modules 1–3 as receptors for the CG base pyr-pur-pyr
type triple helices (Fig. 2).9,10 Prior to incorporation of
each base into oligonucleotides, the ability of 1–3 to
effectively complex the CG base pair in isolation from
the constraints of the DNA duplex was measured
through H NMR studies in an organic solvent. Other
groups have subsequently used this method to evaluate
the hydrogen bonding of other base pair receptors.11
The ureido naphthimidazole base 1 was found to com-
plex CG with an association constant (Kassoc) of
160 Mꢀ1 in CDCl3.9 The complexes of the ureido-
phthalimide base 2 and the ureidoisoindolin-1-one base
3 with CG were found to be more robust with Kassoc
values of 1000 Mꢀ1 and 740 Mꢀ1, respectively.10,12
Unfortunately, the 20-deoxyribose nucleoside derivative
of 2 was found to be unstable to the conditions needed
for incorporation of this base into oligonucleotides.13
Incorporation of a nucleoside analogue of isoindolin-1-
one 3 into a TFO was more successful. Herein we report
the synthesis of a ureido isoindolin-1-one homo-N-
nucleoside 4. This artificial nucleoside was integrated
into oligonucleotide sequences, its ability to bind CG
within a pyr-pur-pyr triple helix was determined using
thermodenaturation studies, and the results compared
to the previously reported host–guest, model studies.
1
The homo-N-nucleoside 4 is an ‘extended nucleoside’
containing an extra methylene groupbetween its
hydrogen bonding, heterocyclic portion and the 20-
deoxyribose moiety. Such homo-N-nucleoside analo-
gues of the natural DNA bases have been investigated
by both Beaucage and Herdwijn.14 To date, base 4 is the
first example of the homo-N-nucleoside design being
employed in a triplex base pair recognition scheme.
However, several of the most promising non-natural
nucleosides designed for recognition of CG and TA
have also possessed modified sugar groups.15 In one of
these examples, Fourrey and Sun’s amide-linked thia-
zole C-nucleoside receptor for TA, an extended base
design was employed similar to the design of 4. The
intervening methylene groupin 4 makes it a longer
nucleoside than the corresponding N-nucleoside 5, pro-
viding 4-CG with improved isomorphism with the nat-
ural base triplets (Fig. 3). A simple two-dimensional
Figure 1. Canonical base triplets in the pyrimidine-purine-pyrimidine
(pyr-pur-pyr) triple helix motif. (R=20-deoxyribose of Watson and
Crick strands and *R=20-deoxyribose of the TFO.)
Figure 3. (a) Two-dimensional overlay of the 4-CG base triplet (red)
with the T-AT base triplet (blue). (b) Two-dimensional overlay of the
N-nucleoside containing 5-CG base triplet (red) with the T-AT (blue).
(R=20-deoxyribose of Watson and Crick strands and *R=20-deoxy-
ribose of the TFO.)
Figure 2. (a) Structures of non-natural receptors designed to form
three hydrogen bonds with the major groove side of the CG base pair.
(b) Isoindolin-1-one homo-N-nucleoside 4 complexed to CG.

E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
1519
Scheme 1.
overlapof the 4-CG triplet with the T-AT base triplet
confirms this assessment as the 20-deoxyribose moiety of
4 extends more closely to the position of the TFO 20-
deoxyribose of a natural base triplet than is possible
with base 5. Simple three-dimensional molecular mod-
eling studies verified the improved isomorphism
obtained by employing the homo-N-nucleoside design.16
confirm the anomeric assignment of 10b (Fig. 4). Thus,
irradiation of H-20 in 10b produced a significant NOE in
only one proton (H-30), while irradiation of H-200 pro-
duced a significant NOE in H-10 only. These results are
consistent with the b-anomer. For comparison, NOE
studies were also performed on the a-anomer 10a, which
was synthesized from 9a using the chemistry described
above. Irradiation of H-20 in 10a gave a network of sig-
nificant NOE values between H-20, H-30, and H-10. This
observation is consistent with the a-anomer as H-30, H-
20, and H-10 all exist on the same face of the furanose
ring.22
2. Results and discussion
The isoindolin-1-one ring system in 4 was constructed
using a key lactamization reaction involving reaction of
4-nitrophenyl 6-(bromomethyl)-3-nitrobenzoate (6) with
aminomethyl-C-glycoside 7 (Scheme 1).17 The base pre-
cursor 6 is available in two steps from methyl 6-methyl-
3-nitrobenzoate,18 and its synthesis has been described
in our previous communication.10 The 20-deoxyribose
derivative 7 was synthesized starting from 30,50-di-O-
toluoyl protected 10-chlorodeoxyribose 8.19 Treatment
of 8 with diethyl aluminum cyanide according to the
method of Beaucage resulted in a 1:1 mixture of nitriles
9a and 9b.14a To obtain the b-anomer of the desired
homo-N-nucleoside, this anomeric mixture of was sepa-
rated by flash chromatography. The a- and b-anomers
were each identified by comparison of previously
1
reported melting point and H NMR data.20 Reduction
of 9b to amine 7 was accomplished using borane-
THF.21
Treatment of 7 with the base precursor 6 and base
resulted in the desired lactamization reaction affording
the isoindolin-1-one derivative 10b. Thorough char-
1
acterization of 10b using H NMR, 13C NMR, COSY,
and HMQC supported formation of the desired lactam.
Difference NOE studies were used to unambiguously
Figure 4. NOE values for the a- and b-anomers of an isoindolin-1-one
homo-N-nucleoside.

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E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
phosphoramidite produced phosphoramidite 14, the
activated starting material for automated oligonucleo-
tide synthesis.23
Phosphoramidite 14 was employed in the automated
synthesis of 15-mer oligonucleotides 15 and 16, each of
which contained the non-natural base in one position.
The crude product of automated DNA synthesis was
analyzed using MALDI-TOF mass spectrometry
(MALDI). The MALDI spectra of 15 and 16 using a
20,40,60-trihydroxyacetophenone (THAP) matrix gave
mass peaks at m/z=4577 and 4609, respectively. These
values were within experimental error of the calculated
molecular weights of 15 (4578) and 16 (4606). Lower
intensity peaks around 2400 were also observed. These
represent the sequence 50-TTTTCTTT-30, which results
from synthetic oligonucleotides which failed to incorpo-
rate phosphoramidite 14. Crude 15 and 16 were purified
using semi-preparative reverse-phase HPLC or denatur-
ing polyacrylamide gel electrophoresis (PAGE).23
Scheme 2.
Coupling MALDI analysis with enzymatic digests of 15
provided complete characterization of the oligonucleo-
tide sequence.24 In one assay, 15 was treated with snake-
venom phosphodiesterase without addition of alkaline
phosphatase. Aliquots of the reaction mixture were
obtained at several time intervals, and each aliquot was
quenched by directly mixing with a 10-fold excess of
THAP matrix. MALDI spectra were obtained of each
aliquot (Fig. 5). Over time, peaks appeared in the MALDI
spectrum corresponding to oligonucleotide fragments
resulting from cleavage of bases from the 30-end of 15.
MALDI analysis of the reaction mixture after 290 min
indicated cleavage upto the point of insertion of the
non-natural base 4. No further cleavage was observed,
The b-homo-N-nucleoside 10b was subjected to Raney
nickel catalyzed hydrogenation to produce amine 11
which was subsequently transformed into the ethyl urea
12 using ethylisocyanate and triethylamine. Removal of
the toluoyl protecting groups with 1% w/v NaOH-
methanol furnished base 4 in good yield.
To incorporate 4 into oligonucleotides, the non-natural
base underwent further manipulations to its 50-O-di-
methoxytrityl (DMT) protected 30-phosphoramidite
(Scheme 2). Thus, reaction of 4 with DMT-Cl installed
the 50-blocking groupin nucleoside 13, and subsequent
treatment of 13 with 2-cyanoethyl diisopropylchloro-
Figure 5. MALDI analysis of digestion of oligonucleotide 15 by snake-venom phosphodiesterase. (a) MALDI spectrum of a digest mixture aliquot
after 24 min. (b) MALDI spectrum of a digest mixture aliquot after 290 min.

E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
1521
suggesting that snake-venom phosphodiesterase has
trouble cleaving past the artificial base.
was next examined. This sequence was designed with
5-methyldeoxycytidine (mC) residues in place of the 20-
deoxycytidine residues. Because the mC-GC base triplet
is more stable than C-GC triplets at pH conditions near
neutrality, it was believed that triple helices containing
16 should be more stable under the thermodenaturation
experiment conditions.26
A similar MALDI-coupled digest experiment was per-
formed using bovine spleen phosphodiesterase, a 50-
exonuclease which sequentially cleaves bases from the
50-end of the oligonucleotide.25 In this case, MALDI
analysis of aliquots from the enzymatic digest mixture
indicated cleavage of bases from the 50-end of 15 upto
and beyond the point of insertion of the artificial
nucleoside 4 (Fig. 6). Taken together, the results of both
the 30-and 50-exonuclease studies strongly indicate that
base 4 is in tact in oligonucleotide 15. Each MALDI
peak observed in the two enzymatic digests is consistent
with this finding. Thus, the possibility that modification
or decomposition of 4 occurred during oligonucleotide
synthesis was ruled out.
Triplex thermodenaturation studies were performed
using TFO 16 and complementary 25-mer duplex tar-
gets of the sequence shown below. Four different 25-mer
targets were studied, each had a different base pair (YZ)
opposite the non-natural base 4.
Oligonucleotide 15 was employed in triple helix ther-
modenaturation (Tm) studies in which the TFO targeted
a homopurine tract within the 25-mer double helix
shown below. Unfortunately, no clear triplex to duplex
transitions were observed in studies employing 15 under
a variety of thermodenaturation conditions.
A triplex dissociation temperature of 18.3 ꢁC was
observed when 4 was opposite CG (Fig. 7). This value
demonstrates that the 4-CG complex within a triple
helix is not robust. By comparison, Tm values of 27.5 ꢁC
and 44.0 ꢁC were recorded when T-AT and C-GC base
triplets replaced the 4-CG base triplet in the identical
triple helix sequence. These canonical base triplets are
thus approximately 1.8–5.0 kcal/mol more stable than
the 4-CG base triplet.27 Moreover, a Tm value of 20.1 ꢁC
was recorded when an abasic site (f) replaced base 4 in
the TFO. This suggests that 4 provided no stabilization
to the triple helix over the abasic site.
One possible method for obtaining sharper triplex to
duplex transitions is enhancing the overall TFO binding
affinity for targeted duplex sequences. Thus, TFO 16
Within the triple helix motifs examined, base 4 exhibited
at best only marginal selectivity in recognizing CG over
the other three base pairs (Table 1). Triple helices iden-
tical to those described above but with GC, AT, and TA
base pairs opposite 4 exhibited Tm values only 0.4 to
1.4 ꢁC lower than the triplex containing the 4-CG base.
This small ÁTm value likely falls within the experi-
mental error of the thermodenaturation studies. The
lack of selectivity for CG is likely a consequence of the
low affinity of 4 for CG within a triple helix.
Figure 7. Melting temperature curve for a triple helix containing the 4-
CG base triplet. A Tm value of 18.3 ꢁC was determined from the first
derivative of this plot (inset).
Figure 6. MALDI analysis of an aliquot taken from a bovine spleen
phosphodiesterase digestion of oligonucleotide 15 obtained after 24 h.

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E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
Table 1. Summary of melting temperature (Tm) values for triple
helices formed from a 15-mer TFO containing base X and a 25-mer
target duplex containing base pairs YZ
Flash chromatography was performed with 32–63
micron silica gel (Merck).30 Radial chromatography was
performed using a Harrison Research Chromatotron on
2 mm plates of silica gel 60 PF254 with gypsum. 1H
NMR, 13C NMR, 31P NMR data were obtained on
either 400 or 500 MHz Varian U400, U500, and 500NB
1
instruments. H NMR spectra obtained in CDCl3 were
Base triplet (X-YZ)
Triple helix Tm (ꢁC)
referenced to 7.26 ppm, and those obtained in CD3OD
were referenced to 3.31 ppm. 13C NMR spectra
obtained in CDCl3 were referenced to 77.23 ppm, and
those obtained in CD3OD were referenced to 49.15
ppm. 31P NMR spectra were referenced to an external
85% aqueous H3PO4 standard. Chemical shifts are
reported in parts per million (ppm) and coupling con-
stants are reported in Hertz (Hz). IR spectra were col-
lected on a Mattson FTIR 5000 with major bands
reported in cmꢀ1. UV/Vis spectra were obtained using a
Shimadzu UV-2501PC recording spectrophotometer.
FDMS and FABMS data were collected by the mass
spectrometry service at the University of Illinois, and
MALDI-TOF spectra were obtained using an Applied
Biosystems Voyager-DE STR mass spectrometer.
MALDI spectra of oligonucleotides were obtained in a
2,4,6-trihydroxyacetophenone (THAP) matrix. Elemental
analysis was performed by the microanalytical labora-
tory at the University of Illinois. Melting points were
measured with a Thomas Hoover melting point appa-
ratus and are uncorrected.
4-CG
4-TA
4-AT
4-GC
f-CG
T-AT
C-GC
18.3
17.2
17.9
16.9
20.1
27.5
44.0
(f=abasic site, see ref. 28 for the C-GC triplet Tm value.)
3. Conclusions
The isoindolin-1-one homo-N-nucleoside 4 was synthe-
sized and successfully incorporated into oligonucleotide
sequences. In the sequence motifs investigated, the 4-
CG base triplet was not robust, and little stabilization
of triplex formation was observed. This result is in con-
trast to our previous finding that the organic-soluble
isoindolin-1-one analogue 3 strongly and effectively
complexed the CG base pair in chloroform.10 Strong
hydrogen bonding in organic solvent thus does not
necessarily translate into effective binding within a
DNA triple helix. Other subtle factors such as base tri-
plet isomorphism, p-stacking, and conformational pre-
organization must also be considered in the design of
nucleosides targeting TA and CG in a triplex-based
recognition scheme. It is possible that the single meth-
ylene groupin 4 between the 20-deoxyribose unit and the
heterocycle is not sufficient to make the 4-CG base tri-
plet isomorphous with T-AT and C-GC. Additionally,
the ability of base 4 to stack with neighboring bases may
be relatively low, thus attenuating any stability that the
4-CG triplet may provide to a triple helix. A productive
avenue may involve retaining the effective hydrogen
bonding motif of base 4 while increasing the tether
between the heterocyclic base and 20-deoxyribose and
improving the ability of the base to stack with neigh-
boring residues. Modifications such as these may ulti-
mately allow the strong hydrogen bonding potential of
the ureido isoindolin-1-one design to result in effective
recognition of CG in a DNA triple helix.29
4.2. Synthesis and characterization of new compounds
4.2.1. 40-Nitrophenyl 6-(bromomethyl)-3-nitrobenzoate
(6). A mixture of 1.00 g (5.60 mmol) N-bromosuccini-
mide, 6.0 mg (0.040 mmol) 2, 20-azobisisobutyronitrile,
0.81 g (2.7 mmol) 4-nitrophenyl-6-methyl-3-nitrobenzo-
ate, and 20 mL benzene was illuminated from below for
5 h with a 100 watt light bulb. During the illumination
process, the reaction mixture heated to reflux. Upon
cooling to 0 ꢁC, a yellow solid precipitated. This solid
was filtered off and discarded. The filtrate was evapo-
rated at reduced pressure to yield an oily orange solid.
This residue was purified using flash chromatography
with a stepped solvent gradient ranging from 1:19 ethyl
acetate–petroleum ether (PE) to 1:4 ethyl acetate-PE to
yield 6 as 0.42 g (41%) of an oily yellow solid containing
a small amount (5% by 1H NMR integration) of
unreacted starting material as an impurity. The product
was further purified to a white solid by precipitation
from 10:1 hexane-ethyl acetate: mp109–111 ꢁC. 1H
NMR (500 MHz, CDCl3) d 9.02 (d, J=2.5, 1H, H-2),
4. Experimental
8.43 (dd, J=8.6, 2.5, 1H, H-4), 8.35 (A, A0 of AA0XX0,
0
0
0
0
4.1. General methods
JAA =2.5, JXX =2.5, JAX=8.0, JAX =0.5, 2H, ArH ),
7.77 (d, J=8.5, 1H, H-5), 7.49 (X,X0 of AA0XX0,
0
0
0
All reactions were performed under N2 in glassware that
was oven dried at 105 ꢁC prior to use. Reagents and
solvents used in reactions were obtained from commer-
cial sources and were used without further purification
except as follows: Tetrahydrofuran (THF) was distilled
from sodium-benzophenone. Pyridine was dried over 4
A molecular sieves. Acetonitrile, benzene, methylene
chloride (CH2Cl2), and triethylamine were distilled from
CaH2. All reactions were monitored by TLC using silica
gel 60 F254 plastic, aluminum, or glass plates (Merck).
JAA =2.5, JXX =2.5, JAX=8.0, JAX =0.5, 2H, ArH),
5.01 (s, 2H, ArCH2Br). 13C NMR (125 MHz, CDCl3) d
162.0, 154.6, 147.3, 146.8, 145.7, 133.2, 128.5, 127.9,
126.8, 126.7, 125.3, 122.49, 28.8. FDMS m/z 382.1
(M+1)+.
4.2.2. N-[(30,50,-O-Ditoluoyl-20-deoxy-ꢀ-D-ribo-pentofur-
anosyl)methyl]-4-nitroisoindolin-1-one (10a). To a solu-
tion of 60 mg K2CO3, 30 mg KI, and 8.0 mL acetonitrile
was added 78 mg (0.20 mmol) (30,50,-O-ditoluoyl-20-

E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
1523
deoxy-a-d-ribo-pentofuranosyl)aminomethane. The resul-
tant solution was stirred in an ice-water bath for 15 min.
A solution of 76 mg (0.20 mmol) 6 in 2 mL acetonitrile
was added, and stirring in an ice-water bath continued
for 5 h. The reaction mixture was filtered through
Celite, and the Celite layer was washed with ethyl ace-
tate. Solvent was evaporated at reduced pressure, and
the remaining brown residue was redissolved in ethyl
acetate. This solution was washed twice with 20 mL
portions of H2O, and four times with 20 mL of satu-
rated aqueous K2CO3. The organic layer was dried over
MgSO4, filtered, and evaporated at reduced pressure to
yield an amber oil. Flash chromatography using a step-
ped solvent gradient ranging from 3:7 ethyl acetate–PE
to 1:1 ethyl acetate–PE afforded 10a as 33 mg (30%) of a
white solid. Compound 10a was isolated at 91% purity,
J=6.0, 2H, ArH), 7.03 (d, J=7.9, 2H, ArH), 5.55 (dt,
J=5.9, 1H, H-30), 4.75 (dd, J=11.9, 2.7, 1H, H-50), 4.67
(B of ABq, JAB=18.9, 1H, H-80), 4.54 (A of ABq,
JAB=18.9, 1H, H-8), 4.54 (m, 1H, H-10), 4.42–4.34 (m,
2H, H-40, H-500), 3.87 (d, J=3.86, 2H, H-60, H-600), 2.42
(s, 3H, ArCH3), 2.35 (dd, J=13.7, 4.6. 1H, H-200), 2.32
(s, 3H, ArCH3), 2.10 (ddd J=13.6, J=11.3, J=6.3, 1H,
H-20). 13C NMR (CDCl3, 125 MHz) d 167.0, 166.5,
166.3, 148.3, 147.9, 144.5, 144.4, 133.8, 129.9, 129.5,
129.3, 127.0, 126.5, 126.3, 123.8, 119.2, 83.6, 79.3, 77.0,
64.8, 52.9, 45.0, 35.9, 21.9, 21.7. IR (film) n 1706.7,
1685.5, 1608.4, 1527. FDMS m/z 544.2 (M+).
4.2.4. N-[(30,-50,-O-Ditoluoyl-20-deoxy-ꢁ-D-ribo-pento-
furanosyl)methyl]-4-amino-isoindolin-1-one (11). To a
solution of 650 mg (1.19 mmol) 10b in 70 mL ethyl
acetate was added 3 mL of a thick suspension of Raney
nickel in ethyl acetate. The resultant mixture was stirred
under 1 atmosphere of hydrogen gas for 23 h. The
reaction mixture was filtered through Celite, and the
Celite layer was washed with 30 mL ethyl acetate.
Combined organic portions were evaporated at reduced
pressure to yield 400 mg (65%) of an oily white solid
which was recrystallized from 1:1 hexane–ethyl acetate
as determined by H NMR integration: mp74–80 ꢁC.
1
1H NMR (CDCl3, 500 MHz) d 8.60 (d, J=2.1, 1H, H-
3), 8.36 (dd, J=8.3, J=2.1, 1H, H-5), 7.88 (d, J=7.9,
2H, ArH0), 7.74 (d, J=7.9, 2H, ArH0), 7.53 (d, J=8.3,
1H, H-6), 7.22 (d, J=7.9, 2H, ArH0), 7.11 (d, J=7.9,
2H, ArH0), 5.45 (dt, J=6.7, J=2.8, 1H, H-30), 4.72 (A of
ABq, JAB=12.1, 1H, H-8), 4.69 (B of ABq, JAB=12.1,
1H, H-80), 4.60 (X of ABX, JAX=2.7, JBX=7.0, 1H, H-
40), 4.57 (X of ABX, JAX=2.7, JBX=7.0, 1H, H-10), 4.52
(A of ABX, JAB=11.6, JAX=2.7, 1H, H-50), 4.43 (B of
ABX, JAB=11.6, JBX=4.1, 1H, H-500), 3.93 (A of ABX,
JAB=14.4, JAX=2.7, 1H, H-60), 3.83 (B of ABX,
JAB=14.4, JBX=7.0, 1H, H-600), 2.73 (dt, J=14.3, 7.6,
1H, H-20), 2.40 (s, 3H, ArCH3), 2.36 (s, 3H, ArCH3),
2.15 (ddd J=14.2, J=5.6, J=3.0, 1H, H-200). 13C NMR
(CDCl3, 125 MHz) d 166.9, 166.5, 166.4, 148.6, 147.9,
144.4, 144.3, 134.2, 129.8, 129.7, 129.4, 129.3, 127.0,
126.8, 126.4, 123.9, 119.5, 82.5, 79.0, 76.8, 64.4, 52.1,
46.8, 35.5, 31.1, 21.9, 21.8. FDMS m/z 544.2 (M+).
to yield 11 as white needles: mp96–99 ꢁC. H NMR
1
(CDCl3, 500 MHz) d 7.90 (d, J=8.1, 2H, ArH0), 7.77 (d,
J=7.9, 2H, ArH0), 7.23 (d, J=7.9, 2H, ArH), 7.09 (d,
J=8.0, 2H, ArH), 7.06 (d, J=1.5, 1H, H-3), 6.96 (d,
J=8.2, 1H, H-6), 6.72 (dd, J=8.2, 2.1, 1H, H-5), 5.48
(d, J=5.6, 1H, H-30), 4.59 (B of ABX, JAB=11.7,
JBX=3.5, 1H, H-500), 4.50 (X of ABX, 1H, JAX=3.3,
JBX=3.5, H-40), 4.48 (A of ABX, JAB=11.7, JAX=3.3,
1H, H-50), 4.39 (s, 2H, H-8, H-80), 4.33 (X of ABX,
JAX=2.2, JBX=5.9, H-10), 3.93 (A of ABX, JAB=14.4,
JAX=2.2, 1H, H-600), 3.81 (br s, 2H, NH2), 3.65 (B of
ABX, JAB=14.4, JBX=5.9, 1H, H-600), 2.40 (s, 3H,
ArCH3), 2.37 (s, 3H, ArCH3), 2.29 (dd, J=13.8, 4.8,
1H, H-200), 2.05 (ddd, J=13.6, 7.5, 4.7, 1H, H-20). 13C
NMR (125 MHz, CDCl3) d 169.4, 166.5, 166.4, 146.7,
144.3, 143.9, 133.5, 131.9, 129.9, 129.6, 129.4, 129.3,
127.1, 126.9, 123.4, 118.9, 108.9, 83.3, 79.3, 76.9, 64.7,
52.2, 45.6, 35.9, 21.8. FDMS m/z 514.3 (M+). Anal.
calcd for C30H30N2O6: C, 70.02; H, 5.88; N, 5.44.
Found: C, 70.14; H, 5.94; N, 5.08.
4.2.3. N-[(30,50,-O-Ditoluoyl-20-deoxy-ꢁ-D-ribo-pentofur-
anosyl)methyl]-4-nitroisoindolin-1-one (10b). To a solu-
tion of 434 mg (3.14 mmol) K2CO3, 199 mg (1.20 mmol)
KI, and 30 mL acetonitrile was added 547 mg (1.43
mmol) 7. The resultant solution was stirred in an ice-
water bath for 15 min. A solution of 800 mg (2.1 mmol)
6 in 30 mL acetonitrile was added slowly dropwise over
15 min. The reaction mixture was allowed to warm to
room temperature, and stirring continued for 4 h. The
reaction mixture was filtered through Celite, and the
Celite layer was washed with ethyl acetate. Solvent was
evaporated at reduced pressure and the remaining
brown residue was redissolved in 50 mL ethyl acetate.
This solution was washed three times with 30 mL por-
tions of H2O and five times with 30 mL portions of
saturated aqueous K2CO3. The organic layer was dried
over MgSO4, filtered, and evaporated at reduced pres-
sure to yield an amber oil. Flash chromatography using
a stepped solvent gradient ranging from 3:7 ethyl ace-
tate–PE to 3:2 ethyl acetate–PE afforded 10b as 535 mg
(69%) of a white solid at >98% purity as determined
4.2.5. N-[(30,-50,-O-Ditoluoyl-20-deoxy-ꢁ-D-ribo-pentofur-
anosyl)methyl]-4-[(ethylamino)-carbonyl]amino-isoindo-
lin-1-one (12). To a solution of 200 mg (0.39 mmol) 11
in 12 mL THF was added 0.20 mL triethylamine and
0.10 mL (1.26 mmol) ethyl isocyanate. The resultant
solution was stirred at 60 ꢁC for 48 h. Solvent was eva-
porated at reduced pressure to yield a white foam. Pur-
ification using flash chromatography with 1:1 ethyl
acetate–CH2Cl2 produced 135 mg (59%) of 12 as a white
solid: mp196–198 ꢁC. H NMR (CDCl3, 400 MHz) d
1
8.06 (dd, J=8.3, 2.1, 1H, H-5), 7.91 (dt, J=8.2, 1.7, 2H,
ArH00), 7.75 (dt, J=8.2, 1.5, 2H, ArH00), 7.58 (br s, 1H,
NH-2), 7.45 (d, J=1.8, 1H, H-3), 7.24 (dd, J=8.6, 0.5,
2H, ArH0), 7.16 (d, J=8.4, 1H, H-6), 7.08 (dd, J=8.6,
0.6, 2H, ArH0), 5.52 (br t, J=6.2, 1H, NH-1), 5.51 (d,
J=4.5, 1H, H-30), 4.66 (dd, J=11.9, 3.34, 1H, H-500),
4.53 (m, 1H, H-10), 4.50 (s, 2H, H-8, H-80), 4.48 (dd,
J=11.9, 4.0, 1H, H-500), 4.36 (m, 1H, H-40), 3.96 (dd,
1
by H NMR integration. A portion of the product was
further purified by recrystallization from 1:1 ethyl ace-
tate–hexane: mp158–160 ꢁC. 1H NMR (CDCl3,
400 MHz) d 8.46 (d, J=1.8, 1H, H-3), 8.20 (dd, J=8.4,
J=2.2, 1H, H-5), 7.92 (d, J=8.4, 2H, ArH0), 7.62 (d,
J=8.3, 2H, ArH0), 7.32 (d, J=8.0, 1H, H-6), 7.25 (d,

1524
E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
J=14.5, 2.5, 1H, H-600), 3.67 (dd, J=14.5, 5.9, 1H, H-
60), 3.34 (qd, J=7.3, 6.9, 2H, CH2), 2.42 (s, 3H,
ArCH3), 2.36 (s, 3H, ArCH3), 2.29 (dd, J=14.0, 5.1,
1H, H-200), 2.09 (ddd, J=13.9, 11.3, 6.4, 1H, H-20), 1.19
(t, J=7.1, 3H, CH3). 13C NMR (CDCl3, 125 MHz) d
169.6, 166.3, 166.2, 156.1, 144.4, 144.6, 134.9, 132.1,
129.8, 129.5, 129.3, 129.2, 126.8, 126.6, 123.4, 122.7,
112.4, 83.3, 78.8, 76.7, 64.5, 52.7, 45.7, 36.1, 34.9, 21.8,
21.7, 15.7. FABMS m/z 586.3 (MH+).
H-50, H-500), 3.75 (s, 7H, OCH3), 3.42–3.14 (m, 5H,
CH2, H-60, H-600 H-40), 2.00–1.81 (m, 2H, H-20, H-200),
1.15 (t, J=7.2, 3H, CH3). 13C NMR (CDCl3, 100 MHz)
d 168.8, 158.3, 155.9, 144.7, 140.3, 135.9, 135.8, 134.1,
131.9, 129.9, 130.0, 129.9, 128.0, 127.7, 122.8, 121.5,
112.9, 111.5, 86.5, 85.9, 77.4, 73.8, 64.4, 55.1, 52.2, 46.8,
38.8, 34.6, 15.4. HRMS (FAB) calcd for C38H41N3O7
(M+) 651.2944, found 651.2943.
4.2.8. N-[50-O-[Bis(400-methoxyphenyl)phenylmethyl]-(20-
deoxy-ꢁ-D-ribo-pentofuranosyl)-methyl]-4-[(ethylamino)-
carbonyl]amino-isoindolin-1-one-30-O-(2-cyanoethyl-N,N0-
diisopropylaminophosporamidite (14). A solution of 109
mg (0.167 mmol) 13, 0.056 mL (0.35 mmol) 2-cya-
noethyl diisopropylchlorophosphoramidite and 0.87 mL
diisopropylethylamine in 1.0 mL CH2Cl2 was stirred at
room temperature for 1.5 h. Methanol was added to the
reaction mixture dropwise and solvent was subsequently
evaporated at reduced pressure to yield a white foam.
Purification using radial chromatography with 1:1 PE–
CH2Cl2 on silica deactivated with triethylamine affor-
ded 91 mg (64%) of a white foam consisting of a mix-
ture of two diastereomers of 14: mp109–116 ꢁC. 1H
NMR (CDCl3, 500 MHz) d 8.28 (dd, J=8.3, 2.1, 1H, H-
5), 8.13 (br s, 1H, NH-2), 7.56 (d, J=1.9, 1H, H-3), 7.41
(m, 2H, ArH), 7.35–7.19 (m, 8H, H6, ArH, ArH, ArH0),
6.78 (d, J=9, 2H, ArH0), 6.76 (d, J=9, 2H, ArH0), 5.94
(br t, J=7.5, 1H, NH-1), 4.60 (A of ABq, JAB=12.4,
1H, H-80), 4.59 (A of ABq JAB=17.4, 1H, H-80), 4.55 (B
of ABq, JAB=12.4., 1H, H-8), 4.53 (B of ABq,
JAB=17.4, 1H, H-8), 4.42 (m, 2H, H30, H10), 4.08 (m,
1H, H-50), 3.95 (dd, J=14.8, 3.1, 1H, H-500), 4.0–3.60
(m, 3H, H-40, OCH2). 3.78 (s, 6H, OCH3), 3.60–3.48 (m,
2H, i-prCH), 3.21–3.09 (m, 2H, H-60, H-600), 2.57 (t,
J=6.7 Hz, 2H, CH2CN), 2.39 (t, J=6.7, 2H, CH2CN),
2.13 (dd, J=12.3, 5.3, 1H, H-200), 2.04 (dd, J=12.3, 5.3,
1H, H200), 1.94–1.82 (m, 1H, H-20), 1.18 (t, J=7.4, 3H,
CH3), 1.15 (d, J=6.8, 6H, i-PrCH3), 1.14 (d, J=6.7,
6H, i-PrCH3), 1.12 (d, J=6.8, 6H, i-PrCH3), 1.04 (d,
J=6.8, 6H, i-PrCH3). 13C NMR (CDCl3, 125 MHz) d
169.6, 158.7, 156.0, 144.9, 140.7, 136.2, 136.1, 135.1,
132.6, 130.3, 128.4, 128.0, 127.0, 123.7, 123.1, 117.7,
116.7, 113.3, 112.8, 86.3, 86.1, 78.2, 75.3, 75.1, 63.9,
58.4, 58.3, 55.4, 52.7, 46.6, 43.4, 43.4, 37.8, 35.0, 24.8,
24.7, 24.6, 20.3, 15.7. 31P NMR (CDCl3, 500 MHz) d
149.1, 148.7. FDMS m/z 852.6 (M+H)+.
4.2.6. N-[(20-Deoxy-ꢁ-D-ribo-pentofuranosyl)methyl]-4-
[(ethylamino)carbonyl]-amino-isoindolin-1-one (4). A solu-
tion of 177 mg (0.30 mmol) 12 in 5 mL 1% w/v NaOH-
methanol was stirred for 1 h. Solvent was evaporated at
reduced pressure with minimal heating to yield a yellow
solid. Purification by flash chromatography with 1:19
methanol-acetone afforded 4 as 87 mg (83%) of an oily
white solid which contained 5–10% residual silica gel as
an impurity: mp 86–93 ꢁC. 1H NMR (CD3OD,
400 MHz) d 7.76 (d, J=2.1, 1H, H-3), 7.60 (dd, J=8.2,
2,1, 1H, H-5), 7.41 (d, J=8.2, 1H, H-6), 4.58 (A of
ABq, JAB=17.8, 1H, H-80), 4.55 (B of ABq, JAB=17.8,
1H, H-8), 4.40 (X of ABX, 1H, JAX=3.2, JBX=7.3, 1H,
H-10), 4.20 (X of ABX, JAX=6.1, JBX=7.0, 1H, H-40), 3.78
(dd, J=8.2, 5.1, 1H, H-30), 3.75 (A of ABX, JAB=11.5,
JAX=3.2, 1H, H-6000), 3.67 (B of ABX, JAB=11.5,
JBX=7.3, 1H, H-60), 3.55 (A of ABX, JAB=11.7,
JAX=6.1, 1H, H-500), 3.52 (B of ABX, JAB=11.7,
JBX=7.0, 1H, H50), 3.23 (q, J=7.2, 2H, CH2), 1.95
(ddd, J=13.0, 5.8, 2.6, 1H, H-200), 1.84 (ddd, J=13.2,
9.3, 6.2, 1H, H-20), 1.15 ( J=7.2, 3H, CH3). 13C NMR
(CD3OD, 125 MHz) d 171.2, 158.2, 141.6, 137.3, 133.9,
124.4, 124.1, 114.2, 89.1, 78.8, 73.7, 63.8, 52.9, 48.0,
39.5, 35.8, 15.8. HRMS (FAB) calcd for C17H23N3O5
(M+H)+ 350.1716, found 350.1714.
4.2.7. N-[50 -O-[Bis(4-methoxyphenyl)phenylmethyl]-(20-
deoxy-ꢁ-D-ribo-pentofuranosyl)methyl]-4-[(ethylamino)-
carbonyl]amino-isoindolin-1-one (13). To a suspension
of 70 mg (0.20 mmol) 4, 4.0 mg 4-(dimethylamino)pyr-
idine, 0.050 mL triethylamine, and 1 mL pyridine was
added 81 mg of 4, 40-dimethoxytrityl chloride. The
resultant clear solution was stirred at room temperature
for 1.5 h. Additional portions of 81 mg 4, 40-dimethoxy-
trityl chloride and 0.050 mL triethylamine were added,
and the reaction mixture was stirred at 45 ꢁC for 13 h.
The reaction mixture was combined with 20 mL H2O
and extracted with 30 mL ethyl acetate. The organic
layer was evaporated at reduced pressure to afford a
yellow oil. Purification using radial chromatography
with a stepped solvent gradient of 1:9 ethyl acetate–
CH2Cl2 to neat ethyl acetate on silica deactivated with
triethylamine afforded 95 mg (72%) of 13 as an oily off-
white solid. The product was isolated at >98% purity
4.3. Synthesis, analysis, and purification of synthetic
oligonucleotides
Solutions of 14 in acetonitrile (1.0 M) were submitted to
the W.M. Keck Center for Comparative and Functional
Genomics (Urbana, IL) for oligonucleotide synthesis on
a 1 mmol scale. Crude products were supplied as lyo-
philized powders. For purification of the crude oligo-
nucleotides, semi-preparative reverse phase HPLC was
performed on a 150 mmꢂ4.6 mm Cosmosil column with
a solvent gradient of 5% acetonitrile–0.10 M triethyl-
ammonium acetate to 25% acetonitrile–0.10 M tri-
ethylammonium acetate at 1.0 mL/min. Fractions
containing the desired oligonucleotide were lyophilized,
and the resulting material was used in Tm studies with-
out further purification. For MALDI-coupled enzymatic
as determined by H NMR integration: mp105–110 ꢁC.
1
1H NMR (CDCl3, 400 MHz) d 8.49 (s, 1H, NH-2), 8.30
(dd, J=8.31, 1.8, 1H, H-5), 7.44 (dd, J=8.1, 1.5, 2H,
ArH), 7.31 (dt, J=8.9, 2.2, 4H, ArH00), 7.29 (d, J=1.9,
1H, H-3), 7.25 (t, J=7.76, 2H, ArH), 7.19–7.15 (m, 1H,
ArH), 7.12 (d, J=8.4, 1H, H6), 6.79 (d, J=9.0, 4H,
ArH0), 6.19 (br t, J=5.0, 1H, NH-1), 4.56–4.26 (m, 2H,
H-10, H-30), 4.49 (A of ABq, JAB=17.3, 1H, H-80), 4.33
(B of ABq, JAB=17.3, 1H, H-800), 4.10–3.94 (m, 2H,

E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
1525
digests, oligonucleotide samples were prepared using
semi-preparative polyacrylamide gel electrophoresis
(PAGE). PAGE was performed on a 0.10 mm 20%
polyacrylamide slab gel prepared with urea. Oligonu-
cleotide samples were loaded as 10 mL of 0.023 mg/mL
solutions in 1:1 H2O–formamide. Electrophoresis pro-
ceeded at 311 V with 1ꢂ tris-Borate as the buffer. The
dominant UV absorbing bands were excised and eluted
into 0.50 M ammonium acetate solutions containing 10
mM magnesium acetate. After filtration, oligonucleo-
tide solutions were desalted using SEP-PAK cartridges
(Waters). Desalted oligonucleotides were eluted from
the cartridges using 6:4 methanol–water, and the resul-
tant solutions were lyophilized.
Microcal. The first derivative of each plot was obtained,
and the local maximum of the first derivative data was
recorded after performance of one smoothing oper-
ation. For all Tm studies, solution temperatures were
measured directly with a Cole-Parmer digital thermistor
temperature gauge. The UV/Vis sample chamber was
continuously flushed with dry nitrogen gas to insure a
low humidity environment, and care was taken to reduce
formation of bubbles in duplex and triplex solutions.
Acknowledgements
We wish to thank the W. M. Keck Center for Com-
parative and Functional Genomics for assistance with
oligonucleotide synthesis. Funding for this work was
generously provided by the University of Illinois and
the National Institutes of Health.
4.4. MALDI-coupled enzymatic digest assays
To 60 mL of a 40 mM solution of TFO 15 in an Eppen-
dorf tube was added 3 mL of a 0.067 mg/mL solution of
snake-venom phosphodiesterase (Sigma). The resulting
solution was thoroughly mixed. Aliquots (1.0 mL) of the
reaction mixture were obtained at regular time intervals
upto 24 h. These aliquots were quenched upon combin-
ation with 5.0 mL of a THAP/ammonium citrate matrix.
Samples (1.0 mL) of the resulting mixture were loaded
directly onto a MALDI plate for analysis.
References and notes
1. (a) Reviews and lead references on triplex DNA: Felsen-
feld, G.; Davies, D. R.; Rich, A. J. Am. Chem. Soc. 1957,
79, 2023. (b) Moser, H. E.; Dervan, P. B. Science 1987,
238, 645. (c) Le Doan, T.; Perrouault, L.; Praseuth, D.;
Habhoub, N.; Decout, J.-L.; Thuong, N. T.; Lhomme, J.;
He
N. T.; He
666. (e) Doronina, S. O.; Behr, J.-P. Chem. Soc. Rev.
1997, 63. (f) Praseuth, D.; Guieysse, A. L.; Helene, C.
Biochim. Biophys. Acta 1999, 1489, 181.
´
le
`
ne, C. Nucleic Acids Res. 1987, 15, 7749. (d) Thuong,
MALDI-coupled spleen phosphodiesterase analysis
proceded in the same manner with the following
modifications: 10 mL of an 0.15 mg/90 mL solution of
calf-spleen phosphodiesterase (Worthington Enzymes;
solution was buffered to pH 6.0 with a 0.15 M ammonium
acetate+0.01 M EDTA buffer) was added to a solution
of 6 mg TFO 15 in 20 mL water+2 mL pH 6.0 ammonium
acetate buffer. The resulting mixture was incubated at
37 ꢁC for 24 h. During this time period, two aliquots
from the mixture were frozen, and the frozen samples
were analyzed using MALDI mass spectrometry.
´
`
lene, C. Angew. Chem., Int. Ed. Engl. 1993, 32,
´
`
2. Maher, L. J., III; Wold, B.; Dervan, P. B. Science 1989,
245, 725.
3. Takasugi, M.; Guendouz, A.; Chassignol, M.; Decout, J.-
L.; Lhomme, J.; Thuong, N. T.; He
Acad. Sci. U.S.A. 1991, 88, 5602.
lene, C. Proc. Natl.
´ `
4. Strobel, S. A.; Dervan, P. B. Science 1990, 249, 73.
5. (a) Reviews of other triplex DNA applications: Maher,
L. J., III Cancer Investigation 1996, 14, 66. (b) Chan, P. P.;
Glazer, P. M. J. Molec. Med. 1997, 75, 267. (c) Malvy, C.,
Harel-Bellan, A., Pritchard, L. L., Eds. Triple Helix
Forming Oligonucleotides; Kluwer Academic Publishers:
Boston, 1999.
6. (a) Mergny, J. L.; Sun, J.-S.; Rougee, M.; Montenay-
Garestier, T.; Barcelo, F.; Chomilier, J.; Helene, C. Bio-
chemistry 1991, 30, 9791. (b) Griffin, L. C.; Kiessling,
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7. Robles, J.; Grandas, A.; Pedroso, E.; Luque, F. J.; Eritja,
R.; Orozco, M. For reviews of non-natural nucleoside
bases for base pair recognition in triple helices see Current
Org. Chem. 2002, 6, 1333. (b) Purwanto, M. G. M.;
Weisz, K. Current Org. Chem. 2003, 7, 427.
8. It should be noted that natural guanosine residues were
found to recognize the TA base pair in the pyr-pur-pyr
triple helix motif: Griffin, L. C.; Dervan, P. B. Science
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9. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc. 1995,
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4.5. UV–Vis triple helix Tm studies
In a typical triple helix thermodenaturation study, a 1.0
mL target duplex solution was prepared by combining a
homopurine strand (0.46–0.5 mM) and
a homo-
pyrimidine strand (0.48–0.92 mM) in a pH 6.5 tris–HCl
melt buffer (0.050 M tris, 0.040 M MgCl2, 0.10 M NaCl,
adjusted to pH 6.5 using concd HCl) at 70 ꢁC.26b This
duplex solution was annealed slowly (cooling rate=1 ꢁC/
min) to 5 ꢁC, and the duplex Tm was determined by
increasing the temperature of the duplex solution (heat-
ing rate=1 ꢁC/min) and observing the absorbance at
260 nm. The temperature of the duplex mixture was
increased until 70 ꢁC. An aliquot of a freshly prepared
aqueous solution of a non-natural base containing TFO
was added to the duplex mixture such that the TFO
concentration was 1.35 mM. The resulting triplex mix-
ture was annealed slowly to 5 ꢁC. The temperature of
the triplex solution was increased at a rate of 1.0 ꢁC/
min. During this process, the absorbance at 260 nm was
observed and recorded at 1 min intervals.
To determine triple helix Tm values, plots of absorbance
at 260 nm vs. temperature were produced in Origin by

1526
E. Mertz et al. / Bioorg. Med. Chem. 12 (2004) 1517–1526
Kuroda, R. Tetrahedron Lett. 1999, 40, 4837. (e) Lopez de
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la Paz, M.; Gonzalez, C.; Vicent, C. Chem. Commun.
2000, 411. (f) Purwanto, M. G. M.; Lengeler, D.; Weisz,
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18. Phadnis, A. P.; Nanda, B.; Patwardhan, S. A.; Gupta,
A. S. Indian J. Chem., Sect. B. 1984, 23, 1098.
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21. Scremin, C. L.; Boal, J. H.; Wilk, A.; Phillips, L. R.;
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22. Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney,
S., IV; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671.
23. Gait, M. J., Ed. Oligonucleotide Synthesis; IRL Press:
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´
12. Complexation of a 4-amino phthalimide base with CG
was also studied in: Lengeler, D.; Weisz, K. Tetrahedron
Lett. 2001, 42, 1479.
13. Zimmerman, S. C.; Mattei, S. Unpublished Results.
14. (a) Boal, J. H; Wilk, A.; Scremin, C. L.; Gray, G. N.;
Phillips, L. R.; Beaucage, S. L. J. Org. Chem. 1996, 61,
8617. (b) Hossain, N.; Blaton, N.; Peeters, O.; Rozenski,
J.; Herdewijn, P. Tetrahedron 1996, 52, 5563.
15. (a) Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T.
Angew. Chem., Int. Ed. 2001, 40. (b) Guianvarc’h, D.;
Benhida, R.; Fourrey, J.-L.; Maurisse, R.; Sun, J.-S.
Chem. Commun. 2001, 1814.
24. Pieles, U.; Zurcher, W.; Schar, M.; Moser, H. E. Nucleic
¨
Acids Res. 1993, 21, 3191.
¨
25. Enzymatic reaction conditions modified from Bernardi,
A.; Bernardi, G. The Enzymes IV, 3rd Ed.; Academic
Press: New York, 1971; p. 329.
26. (a) Lee, J. S.; Woodsworth, M. L.; Latimer, L. J. P.;
Morgan, A. R. Nucleic Acids Res. 1984, 12, 6603. (b)
Conditions for thermodenaturation studies were adapted
from Miller, P. S.; Bhan, P.; Cushman, C. D.; Trapane,
T. L. Biochemistry 1992, 31, 6788.
27. ÁÁG estimates are based on triplex Tm data from Boo-
her, M. A.; Wang, S.; Kool, E. T. Biochemistry 1994, 33,
4645.
28. Zimmerman, S. C.; Schmitt, P. unpublished results.
29. Alternative strategies for sequence specific DNA recogni-
tion have also been developed. Most notable is an
approach involving minor groove recognition by poly-
16. Molecular models of triplex DNA were generated using
(a) Macromodel, version 5.0, produced by Columbia Uni-
versity or (b) Insight II, produced by Accelrys. (c) For
each structure, a natural base was excised from a triplex
structure and a minimized ureido isoindolin-1-one homo-
N-nucleoside structure was placed in the resulting gap. In
the case of models generated by Macromodel, coordinates
were obtained through the Protein Data Bank for an
NMR structure reported in Radhakrishnan, I.; Patel, D. J.
Structure 1994, 2, 17.
17. (a) A related lactamization approach was reported in:
Almansa, C; Gomez, L. A.; Cavalcanti, F. L.; Rodriguez,
´
R.; Carceller, E.; Bartroli, J.; Garcia-Rafanell, J.; Forn, J. J.
Med. Chem. 1993, 36, 2121. (b) Egbertson, M. S.; Naylor,
A. M.; Hartman, G. D.; Cook, J. J.; Gould, R. J.; Hola-
han, M. A.; Lynch, J. J., Jr.; Lynch, R. J.; Stranieri, M. T.;
Vassallo, L. M. Bioorg. Med. Chem. Lett. 1994, 4, 1835.
amides: Dervan, P. B.; Burli, R. W. Current Opinion in
¨
Chemical Biology 1999, 3, 688.
30. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923.