Synthesis of new, base-modified PNA monomers

Article abstract of DOI:10.1080/15257770701490613

A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and 5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling reactions. Starting from the base-unprotected 5-iodo-uracil PNA monomer

Full text of DOI:10.1080/15257770701490613

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Synthesis of New, Base-Modified PNA
Monomers
Ferenc Sipos a & Gyula Sági a
a Institute of Biomolecular Chemistry, Chemical Research Center of
the Hungarian Academy of Sciences , 1025, Budapest, Hungary
Published online: 10 Dec 2007.
To cite this article: Ferenc Sipos & Gyula Sági (2007) Synthesis of New, Base-Modified PNA Monomers,
Nucleosides, Nucleotides and Nucleic Acids, 26:6-7, 681-685, DOI: 10.1080/15257770701490613
To link to this article: http://dx.doi.org/10.1080/15257770701490613
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Nucleosides, Nucleotides, and Nucleic Acids, 26:681–685, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490613
SYNTHESIS OF NEW, BASE-MODIFIED PNA MONOMERS
Ferenc Sipos and Gyula Sa´gi
Institute of Biomolecular Chemistry, Chemical
2
Research Center of the Hungarian Academy of Sciences, 1025 Budapest, Hungary
A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and
2
5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling re-
actions. Starting from the base-unprotected 5-iodo-uracil PNA monomer, only the Stille couplings
were accomplished successfully, while Suzuki couplings with boronic acids containing the same
aryl groups failed. During Sonogashira couplings with terminal alkynes, significant amounts of
unrequired furano[2,3-d]pyrimidine by-products were formed. Protection of the lactam function by
p-methoxybenzylation prevented the opportunity for intramolecular cyclization as well as formation
of a negative charge on the 4-O atom, making it possible to reach almost quantitative yields at
Sonogashira couplings and acceptable conversions in Suzuki reactions.
Keywords Peptide nucleic acids; Pd-catalyzed cross-couplings; 5-substituted-uracils
INTRODUCTION
Peptide nucleic acids (PNAs), containing an N -(2-aminoethyl)glycine
backbone instead of the sugar-phosphate moiety of the natural nucleic
acids, belong to the most powerful and promising DNA mimics due to their
strong hybridization ability with DNA and RNA and complete resistance to
nucleases and proteases.[1] Due to these beneficial properties they are suit-
able for replacement of their corresponding DNA counterparts as therapeu-
tic agents and have proved to be highly efficient diagnostic tools (e.g., PNA
microarrays).
In oligonucleotides 5-C-substitution of uracil base, among other things,
makes possible the introduction of protein recognition elements,[2]
fluorophores,[3] and spin labels.[4] In addition, PNA-DNA chimeras[5] as
well as homogenous oligodeoxynucleotides containing 5-propynyl-uracils
in place of thymines have higher duplex stability[6] and increased mis-
match recognizing ability.[7] The incorporation of different 5-heteroaryl-
uracil moieties into short oligodeoxynucleotides also results in enhanced
Financial support of NKFP MediChem2 (project no. 1/A/005/2004) is gratefully acknowledged.
Address correspondence to Gyula Sa´gi, Institute of Biomolecular Chemistry, Chemical Research
Center of the Hungarian Academy of Sciences, 1025 Budapest, Hungary. E-mail: gsagi@chemres.hu
681

682
F. Sipos and G. Sa´gi
thermal stability of the modified DNA:RNA duplexes compared to that of
the thymine-containing counterpart.[8] This is due to an increased π − π
stacking interaction between the neighboring bases. A similar incorporation
of 5-aryl- and 5-alkynyl-uracils into PNAs is also expected to increase the sta-
bility of PNA:DNA and PNA:RNA complexes, which should also be accom-
panied by a higher mismatch penalty. Some hydrophobic aryl or arylethynyl
substituents are likely to improve the cellular uptake, which is rather poor
in the case of unmodified PNAs.
Syntheses of 5-C-substituted-uracil nucleosides were usually accom-
plished by different Pd-catalyzed cross-coupling (Heck, Sonogashira,
Suzuki, and Stille) reactions, starting from 5-halo-, 5-HgCl-, or 5-OTf-uracil
nucleosides.[9] However, in the synthesis of analogous 5-C-substituted
PNAs, due to different solubility and altered chemical properties (e.g.,
base- and acid-sensitivity) of the N - and C-protected PNA monomers,
it was necessary to change certain reaction conditions and to find the
most convenient starting materials and reagents. Therefore, we aimed
to elaborate on useful synthetic strategies for the preparation of N-Boc
protected PNA monomers containing 5-alkynyl- and 5-aryl-uracil bases, by
application of Pd-catalyzed cross-couplings. In order to study the stabilities
of different PNA:DNA and PNA:RNA complexes incorporation of these
building units into a short (11-mer) homopyrimidine PNA oligomer is in
progress.
RESULTS AND DISCUSSION
Synthesis of Key Intermediates
For the synthesis of 5-C-substituted–uracil PNA monomers by differ-
ent Pd-catalyzed cross-couplings 5-iodo-uracil 1 appeared to be the most
convenient starting material (see Scheme 1). Alkylation of 1 with tert-
butyl-bromoacetate afforded the required N 1-alkyl derivative 2 as a major
product, beside the minor N 3-alkyl- and N 1, N 3-dialkyl compounds. Aci-
dolysis of 2 with TFA followed by coupling of the free acid with N -(2-
Boc-aminoethyl)glycine ethyl ester[10] 3 provided the N 3-unprotected 5-
iodo-uracil PNA monomer 4, which has been synthesized by Hudson et
al.[11] in a similar way. Unfortunately, as it turned out later, starting from
4 only the Stille couplings can be executed successfully, while Suzuki cou-
plings using free aryl-boronic acids failed. In addition, Sonogashira cou-
plings of 4 with terminal alkynes resulted in significant amounts of unre-
quired, ring-closured furano[2,3-d]pyrimidine by-products in all cases. In
order to prevent the opportunity of this intramolecular cyclization, which
is due to the formation of a negative charge on the 4-O-atom in basic
medium, synthesis of an appropriate lactam-protected intermediate was nec-
essary. This was realized by introduction of the orthogonal p -methoxybenzyl

Synthesis of PNA Monomers
683
SCHEME 1 Synthesis of key intermediates.
(PMB) protecting group into the N 3-position of 2. (It must be noted that p -
methoxybenzylation of 4 was not found to be selective for the lactam since
it led to simultaneous benzylation of the Boc-carbamate NH too, even un-
der mild (2–4◦C) conditions.) The N 3-PMB-5-iodo-uracil-1-yl-acetic acid tert-
butyl ester 5 thus obtained was then subjected to acidolysis and coupling to
3 to give the required N 3-protected 5-iodo-uracil analog 6, as an alternative
starting material.
Stille Couplings
Due to lesser electronegativity and thus the stronger metallic character
of tin relative to boron, aryl-trialkylstannanes are better aryl group donors
than the corresponding aryl-boronic acids.[12] Therefore we first tried to
couple the N 3-unprotected 5-iodo-uracil PNA monomer 4 with some aryl-
tributylstannanes in the presence of the Pd(0) catalyst, at elevated tempera-
ture (see Scheme 2). All three reactions proceeded smoothly, leading to the
formation of 5-(2-furanyl-, 2-thienyl-, and phenyl)-uracil PNA monomers 7,
8, and 9, respectively, with good yields. However, since only very few aryl-
trialkylstannanes are commercially available and in addition most of them
are toxic, synthesis of a wide variety of 5-aryl-uracil PNA monomers in order
to carry out studies on alternative methods, such as Suzuki coupling, seemed
reasonable.

684
F. Sipos and G. Sa´gi
SCHEME 2 Synthesis of 5-substituted-uracil PNA monomers.
Suzuki Couplings
Unfortunately, attempted couplings of 4 with the most reactive 2-
thienyl-boronic acid—even despite variation of the solvents (DMF, 1,2-
dimethoxyethane, EtOH), the bases (K2CO3, Cs2CO3, KOt.Bu, NaOEt) and
the Pd-catalysts (Pd(PPh3)4, (PPh3)2PdCl2, Pd(OAc)2 + dppf)—remained
unsuccessful. Since in the presence of a base, due to deprotonation of
the imide NH, there must be a partial negative charge on the 4-O atom,
which is in close proximity with the Pd atom and thus can lower its elec-
trophilic character, thereby the transmetallation step is inhibited. This pre-
sumption was certified by application of the N 3-PMB-protected monomer
6, as the only convenient starting material. Although the isolated yields
were generally lower (37–65%) compared to those of Stille couplings (61–
79%) but the conversions are still acceptable considering that—except for
the 2-thienyl—these substituents are bulkier than aryl groups introduced
by the aryl-tributylstannane reagents. Finally the following N 3-PMB-5-aryl-
uracil PNA monomers were prepared: 5-(2-thienyl) 10, 5-(biphenyl-4-yl) 11,
5-(4-dimethylaminophenyl) 12, and 5-(thianaphten-2-yl) 13. As it was indi-
cated by TLC in the two latter cases the coupling efficiency further increased
when aryl-boronic acid pinacol esters were used instead of the free acids.
Sonogashira Couplings
As previously mentioned, coupling of 4 with three different termi-
nal alkynes gave high overall yields but proportions of the unrequired

Synthesis of PNA Monomers
685
furano[2,3-d]pyrimidine by-products were significantly higher (30–40%)
than those found at analogous couplings (<10%) with sugar-protected 5-
iodo- or 5-TfO-uridines. Although these fluorescent side-products can be
separated completely from the 5-alkynyl-uracil analogs by silica gel chro-
matography but their unexpectedly high proportion still makes it preferable
to start from 6, when in the absence of imide NH intramolecular cycliza-
tions cannot occur. Applying this strategy, the required N 3-PMB-5-alkynyl-
uracil PNA monomers, such as 5-propynyl (14), 5-(1-hexyn-1-yl) 15 and
5-phenylethynyl 16, as sole products, were isolated by nearly quantitative
yields.
UV spectra of all the investigated 5-aryl- and alkynyl-uracil PNA
monomers show large bathochromic shifts relative to that of the thymine-
containing reference compound which refers to a near planar orientation
between the uracil base and the 5-substituents. It is expected to enhance the
base-stacking interactions of adjacent base pairs, leading to higher duplex
stability.
REFERENCES
1. Nielsen, P.E.; Egholm, E.; Berg, E.H.; Buchardt, O. Sequence selective recognition of DNA by strand
displacement with a thymine-substituted polyamide. Science 1991, 254, 1497–1500.
2. Shimkus, M.; Levy, M.; Herman, T.A. Chemically cleavable biotinylated nucleotide: Usefulness in the
recovery of protein-DNA complexes from avidin affinity columns. Proceedings of the National Acadamy
of Sciences U.S.A. 1985, 82, 2593–2597.
3. Crisp, G.T.; Gore, J. Palladium-catalysed attachment of labels with acetylenic linker arms to biologi-
cal molecules. Tetrahedron 1997, 53, 1523–1544.
4. Okamoto, A.; Inasaki, T.; Saito, I. Synthesis and ESR studies of nitronyl nitroxide-tethered
oligodeoxynucleotides. Tetrahedron Lett 2005, 46, 791–795.
5. Bajor, Z.; Sa´gi, Gy.; Tegyey, Zs.; Kraicsovits, F PNA-DNA chimeras containing 5-alkynyl-pyrimidine
PNA units. Synthesis, binding properties and enzymatic stability. Nucleosides, Nucleotides & Nucleic
Acids 2003, 22, 1963–1983.
´
´
¨
6. Sa´gi, J.; Szemzo¨, A.; Ebinger, K.; Szabolcs, A.; Sa´gi, Gy.; Ruff, E.; Otvo¨s, L. Base-modified
oligodeoxynucleotides: Effect of 5-alkyl-, 5-(1-alkenyl)- and 5-(1-alkynyl)-substitution of the pyrim-
idines on duplex stability and hydrophobicity. Tetrahedron Lett 1993, 34, 2191–2194.
7. Barnes III, T.W.; Turner, D.H. Long-range cooperativity in molecular recognition of RNA by
oligodeoxynucleotides with multiple C5-(1-propynyl)pyrimidines. J. Amer. Chem. Soc. 2001, 123,
4107–4118.
8. Gutierrez, A.J.; Terhorst, T.J.; Matteucci, M.; Froehler, B.C. 5-heteroaryl-2ꢁ-deoxyuridine analogs.
Synthesis and incorporation into high-affinity oligonucleotides. J. Amer. Chem. Soc. 1994, 116, 5540–
5544.
9. Agrofoglio, L.A.; Gillaizeau, I.; Saito, Y. Palladium-assisted routes to nucleosides. Chem. Rev. 2003,
103, 1875–1916 and references therein.
10. Breipohl, G.; Will, D.W.; Peyman, A.; Uhlmann, E. Novel synthetic routes to PNA monomers and
PNA-DNA linker molecules. Tetrahedron 1997, 53, 14671–14686.
11. Hudson, R.H.E.; Li, G.; Tse, J. The use of sonogashira coupling for the synthesis of modified uracil
peptide nucleic acid. Tetrahedron Lett 2002, 43, 1381–1386.
12. Yamamoto, Y.; Seko, T.; Namoto, H. New method for the synthesis of boron-10 containing nucleo-
side derivatives for neutron-capture therapy via palladium-catalyzed reaction. J. Organic Chem 1989,
54, 4734–4736.