A novel peptide nucleic acid monomer for recognition of thymine in triple-helix structures

Full text of DOI:10.1021/ja9717424

11116
J. Am. Chem. Soc. 1997, 119, 11116-11117
A Novel Peptide Nucleic Acid Monomer for
Recognition of Thymine in Triple-Helix Structures
Anne B. Eldrup,† Otto Dahl,† and Peter E. Nielsen*,‡
Center for Biomolecular Recognition
Department of Chemistry, UniVersity of Copenhagen
UniVersitetsparken 5
DK-2100 Copenhagen Ø, Denmark
Department of Biochemistry B
The Panum Institute, BlegdamsVej 3
DK-2200 Copenhagen N, Denmark
Figure 1. Proposed structure of the 3-oxo-2,3-dihydropyridazine
E‚T-A triplet.
ReceiVed May 28, 1997
Triplex targeting of double-stranded DNA using natural
nucleobases as introduced ten years ago1 is still essentially
limited to homopurine stretches. The problem lies in the
asymmetric nature of the triple-helix structure, in which only
the purine part of the base pair is recognized by T‚A-T, C+‚G-
C, A‚A-T, or G‚G-C triplet formation.2-4 Attempts to
circumvent this problem by targeting homopurine tracts on
alternate strands5 has expanded the sequence recognition
repertoire somewhat, but naturally has not resulted in a general
solution. It is widely agreed that novel “nucleobases” that are
able to sequence specifically recognize T(-A) and C(-G) base
pairs are required for this task. Several nucleobases that
recognize cytosine in an oligonucleotide context have been
described,6 whereas successful recognition of thymine is still
lacking. Duplex-invading homopyrimidine peptide nucleic acids
(PNAs)7,8 also rely on triplex formation for recognition.9
However, in this case a PNA‚DNA-PNA triplex is formed,
and thus T(or C) recognition by the Hoogsteen PNA strand may
be less stringent, since part of the recognition occurs Via
Watson-Crick base pairing of the other PNA strand. In an
approach to expand the triplex recognition repertoire of PNAs
beyond that of homopurine targets, we now report the synthesis
and recognition properties of a novel “nucleobase”, 3-oxo-2,3-
dihydropyridazine, designed to recognize the T(-A) base(pair).
In order to recognize a T-A base pair from the major groove,
Figure 2. Synthesis of the PNA monomer 4: (a) â-alanine, K2CO3 in
absolute EtOH, reflux overnight, 50%; (b) benzyl alcohol and NaH, 3
h at 165 °C, 57%; (c) ethyl N-(2-Boc-aminoethyl)glycinate, HODhbt
and dicyclohexylcarbodiimide in DMF, overnight at rt, 78%; (d) 1 M
LiOH (aqueous) in THF, 30 min at rt, then 2 M HCl (aqueous), H2,
Pd/C in absolute EtOH, 2 h at 0 °C, 80%.
a heterocycle should be connected to the PNA backbone with
a linker long enough to circumvent the 5-methyl group of
thymine and have a hydrogen donor positioned to bind the 4-oxo
group of thymine. If possible, a hydrogen acceptor to form a
hydrogen bond to one of the N-6 hydrogen atoms of adenine
would also be advantageous. Computer model building indi-
cated that 3-oxo-2,3-dihydropyridazine (E), connected to the
PNA backbone via a â-alanine linker from the 6-position, would
fulfill these requirements (Figure 1). It should also be noted
that the lack of a hydrogen atom on N-1 greatly reduces any
steric interference with the thymine 5-methyl group.
For the synthesis of monomer 4 containing the novel
nucleobase E (Figure 2), commercially available 3,6-dichloro-
pyridazine was converted to N-(3-chloropyridazin-6-yl)-3-
aminopropionic acid (1) by nucleophilic aromatic substitution
with â-alanine in the presence of potassium carbonate. Another
nucleophilic substitution to yield N-(3-(benzyloxy)pyridazin-6-
yl)-3-aminopropionic acid (2) was performed at elevated tem-
perature using benzyl alcoholate in benzyl alcohol. Compound
2 was condensed with ethyl N-(2-Boc-aminoethyl)glycinate10
using the previously published procedure.8 Basic hydrolysis
of the resulting benzyl-protected monomer ester 3, followed by
catalytic hydrogenation of the masked oxo functionality in the
3-position, gave the monomer 4.
† University of Copenhagen.
‡ The Panum Institute.
(1) (a) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-650. (b) Le
Doan, T.; Perrouault, L.; Praseuth, D.; Habhoub, N.; Decout, J. L.; Thuong,
N. T.; Lhomme J.; He´le`ne, C. Nucleic Acids Res. 1987, 15, 7749-7760.
(2) Mergny, J.-L.; Sun, J.-S.; Rougee´, M.; Montenay-Garestier, T.;
Barcelo, F.; Chomilier, J.; He´le`ne, C. Biochemistry 1991, 30, 9791-9798.
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5022.
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2437.
(6) (a) Huang, C.-Y.; Bi, G.; Miller, P. S. Nucleic Acids Res. 1996, 24,
2606-2613. (b) Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc. 1995,
117, 10769-10770. (c) Durland, R. H.; Rao, T. S.; Bodepudi, V.; Seth, D.
M.; Jayaraman, K.; Revankar, G. R. Nucleic Acids Res. 1995, 23, 647-
653. (d) Sasaki, S.; Nakashima, S.; Nagtasugi, F.; Yoshitsugu, T.; Hisatome,
M.; Maeda, M. Tetrahedron Lett. 1995, 36, 9521-9524. (e) Koshlap, K.
M.; Gillespie, P.; Dervan, P. B.; Feigon, J. J. Am. Chem. Soc. 1993, 115,
7908-7909. (f) Stilz, H. U.; Dervan, P. B. Biochemistry 1993, 32, 2177-
2185. Huang, Y.-C.; Cushman, C. D.; Miller, P. S. J. Org.Chem. 1993, 58,
5048-5049. (g) Griffin, L. G.; Kiessling, L. L.; Beal, P. A.; Gillespie, P.;
Dervan, P. B. J. Am. Chem. Soc. 1992, 114, 7976-7982.
(7) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500. (b) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg,
R. H. J. Am. Chem. Soc. 1992, 114, 1895-1897. (c) Egholm, M.; Nielsen,
P. E.; Buchardt, O.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 9677-
9678. (d) Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. 1996, 4, 5-23.
(8) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen,
H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt,
O. J. Org. Chem. 1994, 59, 5767-5773.
In order to assess the triplex recognition properties of the
E-base, monomer 4 was incorporated into the Hoogsteen strand
of a bis-decamer-linked bis-PNA11,12 at two positions facing
(9) (a) Cherny, D. Y.; Belotserkovskii, B. P.; Frank-Kamenetskii, M.
D.; Egholm, M.; Bucharrdt, O.; Berg, R. H.; Nielsen, P. E. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 1667-1670. (b) Nielsen, P. E.; Egholm, M.;
Buchardt, O. J. Mol. Recognit. 1994, 7, 165-170.
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25, 457-461.
S0002-7863(97)01742-3 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 11117
Table 1. Thermal Stabilities of PNA-DNA Complexes
Table 2. Effect of Mismatches on Bis-PNA Complex Stabilities
bis-PNAa
Tm (°C)b
oligonucleotide target sequencea
Tm (°C)b
c
H-TJTETJETTT-eg1-eg1-eg1-TTTACTATCT-NH2
57.0
47.5
5′-dCGCAGATAGTAAACGC-3′
5′-dCGCAGAAAGTAAACGC-3′
5′-dCGCAGAGAGTAAACGC-3′
5′-dCGCAGACAGTAAACGC-3′
5′-dCGCAGAUAGUAAACGC-3′
5′-dCGCAGAAAGUAAACGC-3′
5′-dCGCAGAGAGUAAACGC-3′
5′-dCGCAGACAGUAAACGC-3′
57.0
45.0
40.5
40.5
63.5
49.5
44.5
44.5
H-TJT-aeg(Ac)-TJ-aeg(Ac)-TTT-eg1-eg1-eg1-
d
TTTACTATCT-NH2
H-TJTGTJGTTT-eg1-eg1-eg1-TTTACTATCT-NH2
H-TTT-eg1-eg1-eg1-TTTACTATCT-NH2
H-TTTACTATCT-NH2
46.0
37.0
27.0
42.0
c
H-TJTETJETTT-eg1-eg1-eg1-TTTGCTGTCT-NH2
a Pseudoisocytosine (J) was used instead of protonated cytosine in
the Hoogsteen strand of the bis-PNA. Three units of 8-amino-3,6-
dioxaoctanoic acid (eg1) connect the two antiparallel 10-mer PNA
strands. b Tm measurements were performed in 100 mM NaCl, 1 mM
EDTA, 10 mM Na2HPO4, pH 7.0 on a Gilford Response spectropho-
tometer at a heating rate of 0.5 °C/min. c E is the 3-oxo-2,3-dihy-
dropyridazine unit derived from monomer 4. d aeg(Ac) is N-acetyl-N-
(2-aminoethyl)glycine, a no-base PNA unit.
a The bis-PNA; H-TJTETJETTT-eg1-eg1-eg1-TTTACTATCT-NH2,
was used. b Tm measurements were performed in 100 mM NaCl, 1 mM
EDTA, 10 mM Na2HPO4, pH 7.0, on a Gilford Response spectropho-
tometer at a heating rate of 0.5 °C/min.
The sequence discrimination of this system was studied by
introducing a mismatch at one of the E,A positions. The results
presented in Table 2 show a ∆Tm discrimination in the order of
12-16 °C. Finally, we introduced uracil in the target in place
of thymine in order to assess the interference with the 5-methyl
group. The thermal stability results (Table 2) indicate that the
design of the E-base has not completely solved this steric clash
problem since the E-base PNAs bind somewhat stronger to the
uracil containing targets than to the thymine containing targets
(∆Tm ≈ 3 °C per unit). In conclusion, our results prove the
E‚T-A triplet to be considerably more stable than the G‚T-A
or the no-base triplets, although significantly less stable than
the canonical C+‚G-C and T‚A-T triplets, since complexes
of homopyrimidine decameric bis-PNAs with homopurine DNA
targets typically show thermal stabilities around 70-80 °C. The
results presented herein could direct a novel method for the
future design of heterocycles designed for specific interaction
with T-A base pairs in DNA‚DNA-DNA triplexes.
an adenine in the Watson-Crick strand (Table 1). Thus,
thymines in the DNA target should be recognized at these two
positions. All other positions consisted of T,T and J,C11 pairs
for standard recognition of adenine and guanine in the DNA
target, respectively. In the same system we also substituted
the E-base for a no-base13 position or a guanine.14,15 In an
analogous system we placed a guanine facing the E-base to study
the recognition of cytosine in the DNA target. The thermal
stabilities of complexes of these PNAs with their respective
DNA targets are given in Table 1. These results clearly
demonstrate that the E-base binds stronger to thymine than to
either guanine or a no-base (∆Tm ≈ 5 °C per unit) and also
show that the E-base recognizes thymine over cytosine (∆Tm
≈ 7 °C per unit).
(11) Egholm, M.; Christensen, L.; Dueholm, K.; Buchardt, O.; Coull,
J.; Nielsen P. E. Nucleic Acids Res. 1995, 23, 217-222.
Acknowledgment. This work was supported by the Danish National
Research Foundation. We thank Henrik F. Hansen for fruitful discus-
sions.
(12) PNA oligomerisation was performed as previously described:
Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.;
Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg R. H.
J. Pept. Sci. 1995, 3, 175-183.
Supporting Information Available: Experimental details and
charactarization data for all compounds (3 pages). See any current
masthead page for ordering and Internet access instructions.
(13) L. Christensen and P. E. Nielsen, unpublished results.
(14) Guanine has been shown to interact with thymine to form fairly
stable G‚T-A triplets in DNA‚DNA-DNA triplets2,3 while surprisingly
G‚U-A triplets have been reported to be substantially less stable.15
(15) Miller, P. S.; Cushman, C. D. Biochemistry 1993, 32, 2999-3004.
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