Synthesis and properties of PNA oligomers containing orotic acid derivatives

Article abstract of DOI:10.1081/NCN-200065964

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200065964

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SYNTHESIS AND PROPERTIES OF PNA OLIGOMERS
CONTAINING OROTIC ACID DERIVATIVES
R. H. E. Hudson a & F. Wojciechowski a
a Department of Chemistry , University of Western Ontario , London, Canada
Published online: 15 Nov 2011.
To cite this article: R. H. E. Hudson & F. Wojciechowski (2005) SYNTHESIS AND PROPERTIES OF PNA OLIGOMERS CONTAINING
OROTIC ACID DERIVATIVES, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 1123-1126, DOI: 10.1081/NCN-200065964
To link to this article: http://dx.doi.org/10.1081/NCN-200065964
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1123–1126, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200065964
SYNTHESIS AND PROPERTIES OF PNA OLIGOMERS CONTAINING
OROTIC ACID DERIVATIVES
5
R. H. E. Hudson and F. Wojciechowski
Department of Chemistry, University of
Western Ontario, London, Ontario, Canada
5
We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic
acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA
monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis.
Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic
acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from
the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both
poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were
destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than
modification of an internal position. The type of charge provided by the modification affected the complex
stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.
Modified nucleobases have been intensively studied in the field of nucleic
acids.* Some of our previous work has exploited established chemistry to
incorporate modified nucleobases into PNA.[2] This is desirable as it permits the
incorporation of functional groups without necessarily introducing chirality, and
presents the opportunity to include sites of charge, hydrophilicity, lipophilicity, or
sites for further modification can be distributed along the polymer without
modification of the base-pairing face of the heterocycle.
To our knowledge orotic acid or orotamides have not been incorporated into
oligodeoxynucleotides, thus their effect on PNA structure is speculative. Exami-
nation of molecular models from existing structural information suggests that
PNA:DNA duplexes[3] and PNA:RNA duplexes[4] may tolerate the substitution
better than PNA2:DNA triple-helical complexes,[5] the latter suffering from
unfavorable steric interactions.y However, these three structures also illustrate the
*Reviewed by: Ref. [1].
yHyperChem1 5.1 was used to modify a central thymine or uracil nucleobase in a trimer that was excised
from existing structures. The nucleobase was modified to possess a 6-carboxamide group and was geometrically
optimized using the MM+ force field while the remainder of the structure was held fixed.
Address correspondence to R. H. E. Hudson, Department of Chemistry, University of Western Ontario,
London, Ontario N6A 5B7, Canada.
1123
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1124
R. H. E. Hudson and F. Wojciechowski
SCHEME 1
plasticity of PNA conformation. Therefore, we decided to examine all three types
of complexes, and the results for triple helices are reported herein.
Our approach to the modified PNA was via the methyl orotate monomer
which was synthesized according to Scheme 1. Incorporation of the methyl
orotate monomer (MO) into PNA hexamers was done on NovaSyn TGR resin
(Rink amide) using the FastMoc chemistry module on an Applied Biosystems
433A synthesizer. The amide linkage to the support permits an on-resin
conversion to either an orotamide or orotic acid by the treatment with an amine
or hydroxide prior to liberation of the PNA from the support by acidolysis. For
example, a typical result is illustrated for sequence 5 (Table 1) incorporating the
3-(N,N-dimethylamino)propylamide. This compound was prepared by synthesis of
the sequence AcTTT(MO)TTK-resin followed by treatment with neat 3-(N,N-
dimethylamino)propylamine at 55°C for 6 hr. After cleavage from the resin, the
sequence was isolated as the only significant product (*) by reversed-phase HPLC
(Figure 1, panel A) and identified by mass spectrometry (calcd. for C79H111N29O27
1897.82; obs. 1898.83 MH +, 1920.79 MNa+), Figure 1, panel B.
PNA hexamers bearing a single modification, either at the N-terminus or at an
internal position were synthesized. An unmodified PNA (AcTTTTTTK-NH2,
sequence 1) and a single mismatch sequence (AcTTTCTTK-NH2, not shown) were
used to gauge the tolerance of the complexes to the modified residue. PNAs were
hybridized to poly(rA) or poly(dA) in a 2:1 ratio at a concentration of ꢀ2 mM and
in the presence of 150 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA at pH 7.0. All
compounds were well-behaved and exhibited monophasic cooperative transitions.
Inclusion of a modified residue caused a destabilization of the complexes which
was least severe for the 3-(N,N-dimethylamino)propylamide and most severe for the
orotic acid substituents which indicates that electrostatic effects are important.
However, the substantial loss in complex stability for internal substitution suggests
that the substituents also exert a significant steric effect. The worst tolerated
substituent was found to be the internal orotic acid modification (sequence 8) which
is on par with a complete mismatch (DTm (RNA) = ꢁ43°C).
The methyl orotate monomer is an effective convertible residue compati-
ble with an on-resin conversion strategy. However, in the context of PNA2:NA

R. H. E. Hudson and F. Wojciechowski
1125
TABLE 1
Terminal Modification
Entry
R6
R’6
R’3
R3
Tm (RNA) Tm (DNA) DTm(R) DTm(D)
1
2
H
ꢁCH3 ꢁCH3
H
H
62.0
58.5
57.0
47.2
–
–
H
ꢁCH3
ꢁ3.5
ꢁ9.8
3
4
H
H
ꢁCH3
ꢁCH3
H
H
47.2
46.0
35.8
35.0
ꢁ14.8
ꢁ16.0
ꢁ21.2
ꢁ22.0
Internal Modification
5
H
ꢁCH3
H
34.0
28.0
ꢁ28.0
ꢁ29.0
6
7
H
H
ꢁCH3
ꢁCH3
H
H
22.5
18.3
25.5
24.3
ꢁ39.5
ꢁ41.0
ꢁ31.5
ꢁ32.7
triple-helices this modification is not well tolerated. It should be noted that the
sequences examined contain a modified residue in each PNA domain so its effect
was expected to be large. Currently, we are investigating the duplexes formed
between PNA sequences containing C6-modified uracil and DNA or RNA.
ACKNOWLEDGMENTS
The authors wish to thank NSERC (Canada), the Canadian Foundation for
Innovation, and the University of Western Ontario through the Academic
Development Fund for support of this work.
REFERENCES
1. Revandkar, G.R.; Rao, T.S. DNA with altered bases. In Comprehensive Natural Products: DNA and Aspects of
Molecular Biology, Kool, E.T., Ed.; Elsevier Science Ltd.: Oxford, UK, 2002; Vol. 7, 313–339.
2. Hudson, R.H.E.; Viirre, R.D.; Liu, Y.H.; Wojciechowski, F.; Dambenieks, A.K. Chemistry for the synthesis of
nucleobase-modified peptide nucleic acid. Pure Appl. Chem. 2004, 76(7–8), 1591–1598.

1126
R. H. E. Hudson and F. Wojciechowski
3. Eriksson, M.; Nielsen, P.E. Solution structure of a peptide nucleic acid-DNA duplex. Nat. Struct. Biol. 1996, 3,
410–413.
4. Brown, S.C.; Thomson, S.A.; Veal, J.M.; Davis, D.G. NMR Solution structure of a peptide nucleic acid
complexed with RNA. Science 1994, 265, 777–780.
5. Betts, L.; Josey, J.A.; Veal, J.M.; Jordan, S.R. A nucleic acid triple-helix formed by a peptide nucleic acid-DNA
complex. Science 1995, 270, 1838–1841.