The detrimental effect of orotic acid substitution in the peptide nucleic acid strand on the stability of PNA2:NA triple helices

Article abstract of DOI:10.1139/v05-180

We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hybridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide, significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compatibility of C5 substitution with peptide nucleic acid directed triple helix formation.

Full text of DOI:10.1139/v05-180

1731
The detrimental effect of orotic acid substitution
in the peptide nucleic acid strand on the stability
of PNA2:NA triple helices
Robert H.E. Hudson and Filip Wojciechowski
Abstract: We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid
oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-
based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-
bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or
orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hy-
bridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed
with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by
temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes
formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more
strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the
overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine
PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate
into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide,
significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compati-
bility of C5 substitution with peptide nucleic acid directed triple helix formation.
Key words: peptide nucleic acid, triple helix, orotic acid, orotamide, PNA.
Résumé : On a étudié l’incorporation de dérivés en C6 de l’uracile dans la polypyrimidine, des oligomères du peptide
de l’acide nucléique. Utilisant l’acide uracile-6-carboxylique (acide orotique) comme produit de départ, on a préparé un
monomère compatible avec une synthèse à base de dérivés Fmoc. Ce monomère a ensuite été utilisé comme nucléobase
convertible qui, par traitement des hexamères contenant de l’orotate de méthyle lié à une résine de l’hydroxyde ou par
des amines, conduit à la formation propre d’un peptide de l’acide nucléique qui contient de l’acide orotique ou un oro-
tamide. Les peptides d’hexamères contenant de la nucléobase modifiée par un dérivé en C6 donnent, par le biais de la
formation d’un triplex, une hybridisation tant avec l’acide poly(riboadénylique) qu’avec l’acide poly(désoxyriboadény-
lique). Sur la base des résultats de spectroscopie UV mesurés en fonction de la température, on a observé que les com-
plexes formés à partir de l’acide poly(riboadénylique) sont plus stables que ceux formés à partir de l’acide
poly(désoxyriboadénylique). Toutefois, ces deux complexes sont déstabilisés par rapport aux complexes formés à partir
d’oligomères non modifiés du peptide de l’acide nucléique. Les hexamères internes ou doublement substitués sont en-
core plus déstabilisés que les hexamères substitués en position terminale; de plus, le type de substitution (carboxamide,
ester ou acide carboxylique) affecte aussi la stabilité globale du triplex. Ces résultats montrent clairement que
l’incorporation d’uraciles substitués en C6 dans une polypyrimidine de l’acide polynucléique est nuisible à la formation
de triplex. On a aussi étendu cette chimie à l’introduction d’un uracile 5-carboxylate de méthyle dans un peptide hexa-
mère de l’acide nucléique. Après une conversion sur la résine de l’ester en position C5 en un 3-(N,N-diméthyla-
mino)propylamide, on a observé une stabilisation importante du triplex formé avec de l’acide poly(riboadénylique); ce
qui illustre la compatibilité d’une substitution en C5 avec un peptide de l’acide nucléique en vue de former une hélice
triple.
Mots clés : peptide de l’acide nucléique, triple hélice, acide orotique, orotamide, acide polynucléique.
[Traduit par la Rédaction] Hudson and Wojciechowski 1740
Received 9 May 2005. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 2 December 2005.
R.H.E. Hudson1 and F. Wojciechowski. Department of Chemistry, The University of Western Ontario, London, ON N6A 5B7,
Canada.
1Corresponding author (e-mail: rhhudson@uwo.ca).
Can. J. Chem. 83: 1731–1740 (2005)
doi: 10.1139/V05-180
© 2005 NRC Canada

1732
Can. J. Chem. Vol. 83, 2005
Fig. 1. Structures of DNA and PNA.
Introduction
B
B
B
Modified nucleobases have been intensively studied in the
field of nucleic acids (NAs) for a plethora of reasons (re-
viewed in 1, 2). Inclusion of modified nucleobases in RNA
and DNA provides a route to modulation of the hybridiza-
tion properties (stability, selectivity), artificial expansion of
the genetic code, incorporation of reporter groups, inclusion
of reactive sites for conjugation or further chemistry, and the
ability to undertake fundamental studies on the forces stabi-
lizing nucleic acid complexes, to name but a few current
areas of interest. The study of modified or unnatural nucleo-
bases combined with oligonucleotide analogues containing
backbone structural alterations is a less well-developed area
(3). However, the intersection of these two types of modifi-
cations is fertile ground, in general, for the synthesis and in-
vestigation of oligomers with tunable properties.
In some of our previous work, we have exploited estab-
lished chemistry to incorporate modified nucleobases into
peptide nucleic acid (PNA) (4). Peptide nucleic acid is an
oligonucleotide mimic based on a polyamide backbone,
which is structurally compatible with the formation of heli-
cal complexes with natural DNA or RNA (Fig. 1) (5). Due to
its modular nature, it is relatively straightforward to incorpo-
rate modifications into the N-(2-aminoethyl)glycine back-
bone, the methylene carbonyl linker, or the nucleobase,
either separately or in concert. Thus, the first generation of
peptide nucleic acid is a convenient scaffold for surveying
the effects of changes in these portions of the molecule.
Modification of the nucleobases has the advantage of being
able to introduce a diversity of functional groups (e.g.,
charged, hydrophilic, lipophilic) or sites for conjugation or
further chemistry that do not interfere with the base-pairing
face. Additionally, nucleobase modifications can be done
without introducing chirality. Avoiding changing any of the
-CH2- groups in the backbone and linker region is key to a
modification that circumvents the additional synthetic com-
plexity, challenges to maintaining the integrity of the chiral
center during synthesis/deprotection, and possible conse-
quences to hybridization (6).
The introduction of modified nucleobases into oligo-
nucleotides usually involves time-consuming and laborious
synthesis of a variety of individual nucleobase-modified
monomers each requiring a multistep synthesis. A clever so-
lution to this drawback is to perform a post-synthetic step on
an oligomer containing a convertible nucleobase (7). Herein,
we describe the efficient synthesis of a methyl orotate (MO)
PNA monomer, its smooth incorporation into an oligomer
and straightforward on-resin derivatization to various oligo-
mers. To our knowledge orotic acid or orotamides have not
been incorporated into oligonucleotides, therefore their ef-
fects on PNA cannot be predicted by analogy. To gain some
insight on the possible consequences of exchanging a thy-
mine nucleobase for an orotic acid derivative, a qualitative
molecular modeling exercise was undertaken. Working from
existing structural information, the modeling suggested each
of PNA:DNA duplexes (8), PNA:RNA duplexes (9), and
O
DNA
O
-O
O
O
O
-O
O
O
O
O
P
P
OH
HO
n
B
B
B
O
O
O
PNA
O
O
O
N
N
N
OH
N
H
N
H
H2N
n
PNA2:DNA triple helical complexes (10) could possibly ac-
commodate C6 substitution, although each structure showed
some deformation from the starting conformation.2 Given
the plasticity of PNA conformations, we decided to experi-
mentally investigate all three types of complexes, and the re-
sults for triple helices are reported herein. As a point of
comparison, we have also prepared an oligomer possessing a
carboxamide substitution at C5 of uracil. In DNA chemistry,
substituents at the C5 position of uracil are known to occupy
the major grove and are generally tolerated (2).
Experimental
General
All chemicals were obtained from commercial sources
and were of ACS reagent grade or higher and were used
without further purification, unless noted. Solvents for solu-
tion-phase chemistry were obtained from Caledon Labora-
tories Ltd. (Georgetown, Ontario) and were dried by passing
through columns packed with activated alumina (Innovative
Technology, Inc., Newburyport, Massachusetts). Reagents
for peptide synthesis were obtained from NovaBiochem
(San Diego, California) and synthesis grade solvent from
Caledon Laboratories. 1H NMR spectra were recorded at
400.085 MHz and chemical shifts were referenced to the re-
sidual proton of the deuterated solvent, and 13C NMR spec-
tra were recorded at 100.609 MHz were referenced to the
13C NMR signal of the solvent used. The reported melting
points are uncorrected.
Methyl orotate (2)
Orotic acid monohydrate (24.0 g, 138 mmol) was sus-
pended in methanol (3.0 L) and HCl gas (60 g) was bubbled
into the suspension. Reflux was maintained for 24 h after
which time the suspension had cleared. The volume was re-
duced by two-thirds, and cooled for 5 h below 0 °C. The
white crystals were collected by filtration and washed with
cold methanol to yield 21.3 g (91%). TLC (EtOAc–MeOH,
80:20): Rf 0.58; mp 249–251 °C. 1H NMR (400 MHz,
DMSO, ppm) δ: 11.40 (s, 1H), 11.16 (s, 1H), 6.03 (s, 1H),
3.83 (s, 3H). 13C NMR (100 MHz, DMSO, ppm) δ: 163.5,
160.5, 150.5, 141.1, 103.5, 53.4.
2 HyperChem 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 remain-
der of the structure was held fixed.
© 2005 NRC Canada

Hudson and Wojciechowski
1733
7.5 Hz), 6.09 (br s, 1H), 5.05 (s, ma., 1.2H) and 4.86 (s, mi.,
0.8H), 4.36–4.18 (m) and 3.90 (s, ma.) (5H), 3.75 (s, 3H),
3.39–3.07 (m, 4H). 13C NMR (100.6 MHz, DMSO-d6, ppm)
δ: 167.9, 167.4, 162.2, 161.6, 156.3, 151.1, 144.7, 143.9,
140.8, 127.6, 127.1, 125.1, 120.1, 80.9, 65.5, 53.5, 48.9,
46.8, 44.6, 41.8, 27.7. HR-MS (ESI-TOF) calcd. for sodium
adduct C31H34N4O9Na: 629.2225; found: 629.2217.
tert-Butyl (methyl orotate-1-yl)acetate (3)
Methyl orotate (18.9 g, 111.0 mmol) was suspended in
dry DMF (600 mL) and K2CO3 (15.34 g, 111.0 mmol) was
added and the mixture was stirred under N2 at room temperature
for 30 min. tert-Butyl bromoacetate (18.0 mL, 122.0 mmol)
was added dropwise over 1 h and the suspension was vigor-
ously stirred for 48 h under an atmosphere of nitrogen. The
mixture was filtered and solvent removed in vacuo, the resi-
due was extracted with ethyl acetate (4 × 250 mL) and water
(100 mL). The combined organic phase was washed with
brine, dried (Na2SO4), filtered, and concentrated in vacuo.
The material contained a small amount of dialkylated
product, which could be removed by recrystallization from
absolute ethanol to yield 19.3 g (61%) of a white solid. TLC
N-[2-Fmoc-aminoethyl]-N-[(methyl orotate-1-
yl)acetyl]glycine (6)
Compound 5 (5.20 g, 8.57 mmol) was suspended in
30 mL of CH2Cl2 and cooled to 0 °C and the mixture was
stirred under nitrogen, and Et3SiH (1.37 mL, 8.57 mmol)
was added. TFA (30 mL) was added dropwise over 30 min
while stirring on ice under an atmosphere of nitrogen. The
solution was stirred for an additional hour while in the ice
bath and then allowed to warm to room temperature while
stirring was continued for an additional 4 h, after which
TLC analysis showed complete consumption of the starting
material. The mixture was evaporated to dryness in vacuo
and the remaining volatiles were removed by repeated co-
evaporation with CH2Cl2 and diethyl ether. The white solid
was triturated with a mixture of 30 mL of CH2Cl2 and
60 mL of diethyl ether and cooled below 0 °C for 12 h. The
product was isolated by filtration to give 3.87 g (82%) of a
white solid. TLC (EtOAc–MeOH–AcOH, 80:15:5): Rf 0.30;
1
(EtOAc–MeOH, 90:10): Rf 0.76; mp 186–188 °C. H NMR
(400 MHz, DMSO, ppm) δ: 11.85 (s, 1H), 6.13 (s, 1H), 4.60
(s, 2H), 3.80 (s, 3H), 1.40 (s, 9H). 13C NMR (100 MHz,
DMSO, ppm) δ: 167.1, 162.2, 161.5, 151.0, 143.8, 105.6,
81.8, 53.7, 46.2, 27.6. HR-MS (EI) calcd. for C12H16N2O6:
284.1009; found: 284.1009.
(Methyl orotate-1-yl)acetic acid (4)
Compound 3 (7.0 g, 24.6 mmol) was suspended in 80 mL
of CH2Cl2 and cooled to 0 °C, the mixture was stirred under
nitrogen, and Et3SiH (3.9 mL, 24.4 mmol) was added. TFA
(80 mL) was added dropwise over 30 min, with stirring un-
der nitrogen. The solution was stirred for 1 h while in an ice
bath and then allowed to warm to room temperature for 3 h.
The mixture was evaporated to dryness in vacuo and the
remaining volatiles were removed by successive co-
evaporations with CH2Cl2 (3×) and diethyl ether (1×). The
white solid was triturated with a mixture of 30 mL of
CH2Cl2 and 60 mL of diethyl ether and cooled below 0 °C
for 12 h; filtration gave 5.04 g (89%) of a white solid. TLC
(EtOAc–MeOH–AcOH, 80:15:5): Rf 0.35; mp 206 to
1
mp (dec.) 157 to 158 °C. H NMR (400 MHz, DMSO, ppm)
δ: 11.81 (br s, 1H), 7.89 (d, 2H, J = 7.4 Hz), 7.66 (d, 2H,
J = 6.8 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.32 (t, 2H, J =
7.4 Hz), 6.07 (br s, 1H), 5.05 (s, ma., 1.2H) and 4.86 (s, mi.,
0.8H), 4.34–4.20 (m) and 3.93 (s, ma.) (5H), 3.74 (s, 3H),
3.37–3.08 (m, 4H). 13C NMR (100 MHz, DMSO, ppm) δ:
170.4, 167.4, 162.3, 161.7, 156.5, 151.2, 144.8, 144.0,
140.8, 127.7, 127.1, 125.2, 120.2, 105.2, 65.6, 53.6, 48.0,
47.1, 46.8, 45.0, 44.6. HR-MS (ESI-TOF) calcd. for sodium
adduct C27H26N4O9Na: 573.1599; found: 573.1595.
1
207 °C. H NMR (400 MHz, DMSO, ppm) δ: 11.84 (s, 1H),
6.13 (s, 1H), 4.62 (s, 2H), 3.80 (s, 3H). 13C NMR
(100 MHz, DMSO, ppm) δ: 169.6, 162.3, 161.6, 151.5,
144.0, 105.5, 53.70, 45.8. HR-MS (EI) calcd. for
C12H8N2O6: 228.0382; found: 228.0377.
5-Hydroxymethyluracil (7)
Triethylamine (42.0 mL, 0.30 mol) was added to a sus-
pension of uracil (22.4 g, 0.20 mol) and paraformaldehyde
(18.0 g, 0.60 mol) in 600 mL of water. The solution turned
clear when heated to 65 °C and was allowed to stir for 12 h
at 65 °C. The water was removed under vacuum until ap-
proximately 50 mL of volume remained. To the residue was
added 60 mL of 95% ethanol and the solution was allowed
to stand for 30 min at room temperature and further cooled
at 4 °C. The white precipitate was collected by filtration and
was washed with 95% ethanol. The procedure of concentrat-
ing the filtrate and precipitation with 95% ethanol was re-
peated three times and the combined mass was 25.3 g (89%)
of a white solid. TLC (CHCl3–MeOH, 80:20): Rf 0.15; mp
tert-Butyl N-[2-Fmoc-aminoethyl]-N-[(methyl orotate-1-
yl)acetyl]glycinate (5)
Compound 4 (4.0 g, 17.5 mmol) and HOBt hydrate
(2.68 g, 17.53 mmol) were dissolved in 20 mL DMF and
stirred for 10 min while kept under a nitrogen atmosphere.
DCC (3.61 g, 17.5 mmol) was added and the mixture was
stirred on ice for 30 min. The free Fmoc-backbone tert-butyl
ester (2) (6.95 g, 17.53 mmol) was dissolved in 5 mL
CH2Cl2 and added to the mixture. The reaction mixture was
stirred for 24 h at room temperature, extracted with EtOAc
(4 × 150 mL) and phosphate buffer (pH ~ 4.5, 2 × 50 mL),
and the combined organic phase was washed with sodium
bicarbonate (2 × 50 mL) and brine (50 mL), dried (Na2SO4),
filtered, and concentrated in vacuo. The solid was
recrystallized from a mixture of 10 mL of MeOH and 25 mL
of isopropanol, and isolated by filtration to give 7.44 g
(70%) of a white solid. TLC (EtOAc–MeOH, 90:10): Rf
1
(dec.) 360 °C. H NMR (400 MHz, DMSO, ppm) δ: 11.06
(s, 1H), 10.71 (s, 1H), 7.23 (s, 1H), 4.84 (s, 1H), 4.08 (s,
2H). 13C NMR (100 MHz, DMSO, ppm) δ: 163.0, 151.5,
138.5, 112.8, 56.0.
5-Formyluracil (8)
Hydroxymethyluracil (21.50 g, 151.30 mmol) was dis-
solved in 800 mL of water by heating to approximately
90 °C. The solution was then cooled to 45 °C, K2S2O8
(75.0 g, 277.4 mmol) and AgNO3 (0.80 g, 4.71 mmol) were
1
0.74; mp (dec.) 171–173 °C. H NMR (400 MHz, DMSO-
d6, ppm) δ: 11.79 (br s, 1H), 7.88 (d, 2H, J = 7.4 Hz), 7.66
(d, 2H, J = 6.9 Hz), 7.40 (t, 2H, J = 7.3 Hz), 7.31 (t, 2H, J =
© 2005 NRC Canada

1734
Can. J. Chem. Vol. 83, 2005
added, and the product began to slowly precipitate. The re-
action was stirred for a further 20 min at 40 °C and then
cooled to room temperature over 15 min while stirring was
continued. The suspension was then placed at 4 °C, the off-
white crystals were collected by filtration, and rinsed with
50 mL of cold water to yield 17.65 g (83%) of the title com-
DMSO, ppm) δ: 11.79 (s, 1H), 8.59 (s, 1H), 4.53 (s, 2H),
3.71 (s, 3H), 1.40 (s, 9H). 13C NMR (100 MHz, CHCl3,
ppm) δ: 165.6, 163.2, 158.8, 152.3, 149.6, 105.3, 84.1, 52.5,
49.9, 28.0. HR-MS (EI) calcd. for C12H16N2O6: 284.1009;
found: 284.1009.
1
pound; mp 302–304 °C. H NMR (400 MHz, DMSO, ppm)
(Uracil-5-methylcarboxylate-1-yl)acetic acid (12)
δ: 11.89 (s, 1H), 11.49 (s, 1H), 9.72 (s, 1H), 8.12 (s, 1H).
13C NMR (100 MHz, DMSO, ppm) δ: 186.8, 162.6, 150.6,
149.4, 110.2.
Compound 11 (1.61 g, 5.66 mmol) and Et3SiH (0.75 mL,
4.70 mmol) were suspended in 20 mL DCM, cooled to 0 °C,
and 15 mL of TFA was added dropwise. The reaction was
stirred for 1 h on ice and an additional 2.5 h at room temper-
ature. The mixture was evaporated to dryness in vacuo and
the remaining volatiles were removed by co-evaporation
with CH2Cl2 and diethyl ether. The compound was triturated
with a mixture of approximately 5 mL of CH2Cl2 and 80 mL
of diethyl ether and cooled to 0 °C overnight. Filtration and
drying under vacuum gave 1.22 g (94%) of a white solid.
TLC (EtOAc–MeOH–AcOH, 80:15:5): Rf 0.16; mp 227–
Uracil-5-carboxylic acid (9)
5-Formyluracil (4.30 g, 30.70 mmol) was dissolved in
350 mL of water by heating to reflux. To this solution was
added K2S2O8 (8.30 g, 30.70 mmol) and AgNO3 (50 mg,
0.29 mmol), and the reaction was stirred while cooling to
65 °C over 30 min. A precipitate formed upon concentrating
the volume to 50 mL. This mixture was cooled to 4 °C to
promote the precipitation, and the product was subsequently
collected by filtration. The title compound was purified by
suspending it in 125 mL of water, adding 4 mol/L NaOH un-
til a clear solution was obtained, and reprecipitating the
compound by dropwise addition of concd. HCl until pH 2.0.
Cooling the mixture to 4 °C followed by filtration yielded
1
229 °C. H NMR (400 MHz, DMSO, ppm) δ: 11.68 (s, 1H),
8.59 (s, 1H), 4.55 (s, 2H), 3.70 (s, 3H). 13C NMR
(100 MHz, CHCl3, ppm) δ: 169.2, 163.0, 159.5, 153.4,
150.2, 103.4, 51.6, 49.2. HR-MS (EI) calcd. for C12H8N2O6:
228.0382; found: 228.0375.
1
3.34 g (70%) of a white solid; mp (dec.) 286–288 °C. H
tert-Butyl N-[2-Fmoc-aminoethyl]-N-[(uracil-5-
NMR (400 MHz, DMSO, ppm) δ: 12.73 (s, 1H), 11.99 (s,
2H), 8.25 (s, 1H). 13C NMR (100 MHz, DMSO, ppm) δ:
165.2, 163.5, 150.4, 150.2, 101.2.
methylcarboxylate-1-yl)acetyl]glycinate (13)
Compound 12 (0.210 g, 0.92 mmol), HOBt hydrate
(0.141 g, 0.92 mmol), and DCC (0.190 g, 0.92 mmol) were
dissolved in 3 mL of DMF and stirred under nitrogen for
15 min. The free amine form of the Fmoc-backbone tert-
butyl ester (0.365 g, 0.92 mmol) was dissolved in a minimal
amount of DMF and added to the mixture along with
Hünings base (0.169 mL, 0.97 mmol). The reaction was
stirred for 24 h at room temperature and extracted with
EtOAc, sodium bicarbonate, phosphate buffer (pH ~ 4.5),
and brine. The combined organic phases were dried
(Na2SO4), filtered, and concentrated in vacuo. The solid was
purified by flash chromatography (DCM–MeOH gradient) to
yield 0.298 g (55%) of a white solid. TLC (EtOAc–MeOH,
Uracil-5-methylcarboxylate (10)
Uracil-5-carboxylic acid (2.50 g, 16.00 mmol) was sus-
pended in methanol (150 mL) and HCl gas was bubbled into
the suspension. The HCl was generated by the dropwise
combination of 40 mL of concd. H2SO4 and 40 mL of
concd. HCl. Once the mixture was saturated with HCl, it
was refluxed for 12 h and then the solvent was removed in
vacuo. The product was then recystallized from methanol to
yield 2.34 g (86%) of a white solid. TLC (EtOAc–MeOH,
90:10): Rf 0.52; mp (dec.) 267 to 268 °C. 1H NMR
(400 MHz, DMSO, ppm) δ: 11.61 (s, 1H), 11.31 (s, 1H),
8.12 (s, 1H), 3.67 (s, 3H). 13C NMR (100 MHz, DMSO,
ppm) δ: 163.2, 160.1, 150.6, 149.7, 102.9, 51.4. HR-MS
(EI) calcd. for C6H6N2O4: 170.0328; found: 170.0324.
1
90:10): Rf 0.67; mp (dec.) 135–137 °C. H NMR (400 MHz,
DMSO, ppm) δ: 8.43 (s, ma., 0.6H), 8.34 (s, mi., 0.4H),
7.89 (d, 2H, J = 7.4 Hz), 7.68 (m, 2H), 7.40 (t, 2H, J =
7.4 Hz), 7.32 (t, 2H, J = 7.4 Hz), 7.24 (m, 1H), 4.85 (s, ma.,
1.2H) and 4.86 (s, mi., 0.8H), 4.34–4.16 (m) and 3.98 (s,
ma.) (5H), 3.68 (s, 3H), 3.28–3.02 (m, 4H). 13C NMR
(100 MHz, DMSO, ppm) δ: 167.9, 166.8, 163.0, 159.5,
156.1, 153.7, 150.1, 143.9, 140.8, 127.6, 127.1, 125.1,
120.1, 103.2, 81.0, 65.5, 51.5, 50.0, 48.8, 48.3, 46.7, 27.7.
HR-MS (ESI-TOF) calcd. for sodium adduct C31H34N4O9Na:
629.2225; found: 629.2254.
tert-Butyl (uracil-5-methylcarboxylate-1-yl)acetate (11)
Uracil-5-methylcarboxylate (2.06 g, 12.11 mmol) was dis-
solved in dry DMF (40 mL), and K2CO3 (1.67 g,
12.11 mmol) was added. tert-Butyl bromoacetate (1.79 mL,
12.11 mmol) was added dropwise to the stirred mixture and
stirring was continued for 24 h while the reaction mixture
was kept under an atmosphere of nitrogen. The reaction mix-
ture was filtered to remove the undissolved potassium car-
bonate and then the solvent was removed in vacuo. The
residue was extracted with ethyl acetate (3 × 75 mL) and
water (25 mL). The combined organic layers were then
washed with brine, dried (Na2SO4), filtered, and concen-
trated in vacuo. The crude product was recrystallized from
methanol to yield 2.14 g (62%) of a white solid. TLC
(EtOAc–MeOH, 90:10): Rf 0.71; mp 179 to 180 °C. 1H
NMR (400 MHz, CHCl3, ppm) δ: 8.69 (s, 1H), 8.19 (s, 1H),
N-[2-Fmoc-aminoethyl]-N-[(uracil-5-methylcarboxylate-
1-yl)acetyl]glycine (14)
Compound 13 (0.250 g, 0.41 mmol) and Et3SiH (59.0 uL,
0.37 mmol) were suspended in 2 mL DCM, cooled to 0 °C,
3 mL of TFA was added dropwise, and the reaction was
stirred for 1 h in an ice bath and 2.0 h at room temperature.
The mixture was evaporated to dryness in vacuo and the re-
maining volatiles were removed by co-evaporation sequen-
tially with CH2Cl2 and diethyl ether. The solid was slurried
in CH2Cl2–diethyl ether, cooled to 0 °C for 12 h, and subse-
1
4.43 (s, 2H), 3.86 (s, 3H), 1.47 (s, 9H). H NMR (400 MHz,
© 2005 NRC Canada

Hudson and Wojciechowski
1735
Table 1. Mass spectral characterization of PNA oligomers.
Found
Calcd.
Entry
Formula
[M + H]+
M+
[M + H]+
M+
Method
Resolution
7
C74H101N27O26
C75H101N27O28
C75H101N27O28
C76H101N27O30
C74H99N27O28
C74H99N27O28
C74H97N27O30
C79H111N29O27
C79H111N29O27
C84H121N31O28
C76H104N28O28
1784.6012
1784.7489
MALDI
ESI
M
A
8
1829.25
1829.10
1828.79
1828.79
9
ESI
A
10
11
12
13
14
15
16
17
1872.7588
1872.7286
MALDI
ESI
M
A
1814.69
1814.65
1814.77
1814.77
ESI
A
1844.5623
1898.8276
1844.6973
1898.8283
MALDI
ESI
M
M
A
1899.14
1858.14
1898.93
1857.84
ESI
2012.7640
ND
2012.9076
ND
MALDI
ESI
M
A
18
19
1828.59
1898.63
1828.79
1898.93
ESI
ESI
A
A
C75H101N27O28
C79H111N29O27
20
Note: Abbreviations in use: monoisotopic mass determination (M), average mass determination (A), and not determined (ND).
quently filtered to give 0.187 g (83%) of a white solid. TLC
30 min. The resin was transferred to a peptide vessel, rinsed
with H2O, THF, and DCM. The resin was dried in vacuo
and cleavage and release of the orotic-acid-containing oligo-
mers was performed as previously described.
(EtOAc–MeOH–AcOH, 80:15:5): Rf 0.11; mp (dec.) 146 to
1
147 °C. H NMR (400 MHz, DMSO, ppm) δ: 8.43 (s, ma.,
0.6H), 8.35 (s, mi., 0.4H), 7.89 (d, 2H, J = 7.3 Hz), 7.68 (d,
2H, J = 7.7 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.32 (t, 2H, J =
7.3 Hz), 4.86 (s, ma., 1.0H) and 4.86 (s, mi., 0.6H), 4.33–
4.17 (m) and 3.98 (s, ma.) (5H), 3.68 (s, 3H), 3.30–3.09 (m,
4H). 13C NMR (100 MHz, DMSO, ppm) δ: 170.33, 166.79,
162.99, 159.52, 156.35, 153.67, 150.06, 143.87, 140.73,
127.61, 127.07, 125.12, 120.11, 103.18, 65.49, 51.53, 48.31,
47.78, 46.88, 46.72, 46.22. HR-MS (ESI-TOF) calcd. for so-
dium adduct C27H26N4O9Na: 573.1599; found: 573.1597.
On resin aminolysis of oligomers containing C5 or C6
esters
To 6 mg (~1 µmol) of the appropriate resin bound
oligomer (1–3, 19), in a glass vial, was added 1 mL of the
appropriate amine, the vial was purged with nitrogen,
capped and allowed to stir for 6 h at 55 °C. The resin was
transferred to a peptide vessel and rinsed with DCM. Cleav-
age and release of the orotamide-containing oligomers was
performed as previously described.
Synthesis of PNA oligomers
PNA hexamers were synthesized on a 5 or 10 µmol scale
using the standard FastMoc module on an Applied Bio-
systems 433A synthesizer employing NovaSyn TGR (Rink
amide) resin.
Purification of PNAs
Crude PNA, isolated as previously described, was
redissolved in 0.1% TFA in water (approximately 4 mg/mL)
and purified by reversed phase HPLC using a 250 mm ×
4.6 mm, C18-bonded phase, 100 pore, 5 µm particle size
column. A binary gradient elution was used 0–5 min, 100%
A; 5–20 min, 100% to 80% A; 20–40 min, 80% to 70% A,
wherein solvent A is 0.1% TFA in water and solvent B is
0.1% TFA in MeCN.
Cleavage from resin
To 6 mg (~1 µmol) of the appropriate resin-bound oligo-
mer (8–10), in a peptide vessel, was added a 1 mL solution
containing ice-cold TFA–Et3SiH (0.975:0.025), the vessel
was purged with nitrogen and the mixture was shaken for
2 h. The solution was emptied into a 5 mL flask and the
resin was rinsed with 0.5 mL of TFA. The combined TFA
was evaporated with a stream of nitrogen and 3 mL of cold
ether was added. Upon 1 h of cooling, the ether was de-
canted and a white precipitate was collected. The solid was
dissolved in 0.1% TFA–H2O, filtered, and purified under
RP-HPLC.
Characterization of base-modified PNAs
Mass spectral analysis of oligomers was done at the Dr.
Don Rix Protein Identification and UWO Biological Mass
Spectrometry Laboratory. Electrospray ionization time-of-
flight mass spectrometry (ESI-TOF MS) was done with mass
calibration provided by Glu-fibrinopeptide 1 (m/z (+2) =
785.842, monoisotopic mass), myoglobin (M+ = 16951.499,
average mass), or histatin 5 (M+ = 3034.515, monoisotopic
mass). Matrix-asisisted laser desorption ionization time-of-
flight mass spectrometry (MALDI-TOF MS) was performed
using α-cyano-4-hydroxycinnamic acid as the matrix, and
calibration for monoisotopic masses was provided by angio-
Hydrolysis of methyl orotate oligomers
To 6 mg (~1 µmol) of the appropriate resin-bound oligo-
mer (8–10), in a microfuge tube (Eppendorf), was added
50 µL of THF and 100 µL of 1 mol/L NaOH. The tubes were
allowed to stand for 30 min and heated to 50 °C for a further
© 2005 NRC Canada

1736
Can. J. Chem. Vol. 83, 2005
Scheme 1. Reagents and conditions: (a) HCl(g)–MeOH, reflux for 24 h; (b) K2CO3, DMF, BrCH2CO2-t-Bu, 24 h; (c) CF3COOH,
DCM, Et3SiH, 1 h at 0 °C, 3 h at room temperature.
O
O
O
O
N
NH
NH
NH
NH
a, 91%
b, 61%
c, 89%
HO
O
O
O
N
H
O
N
H
O
N
O
O
O
O
O
O
O
O
O
OH
4
3
2
1
Scheme 2. Reagents and conditions: (a) (HCHO)n, 0.5 mol/L Et3N, H2O, 12 h at 65 °C; (b) K2S2O8, AgNO3, H2O; (c) K2S2O8,
AgNO3, H2O; (d) HCl(g)–MeOH, reflux for 12 h; (e) K2CO3, DMF, BrCH2CO2-t-Bu, 24 h; (f) CF3COOH, DCM, Et3SiH, 1 h at 0 °C,
2.5 h at room temperature.
O
O
O
O
O
O
NH
H
NH
HO
NH
HO
NH
c, 70%
b, 83%
a, 89%
N
H
O
N
H
O
N
H
O
N
H
O
7
8
9
d, 86%
O
O
N
O
O
N
O
O
O
NH
O
NH
e, 62%
f, 94%
O
NH
O
O
O
N
H
O
O
12
11
10
OH
O
tensin I ([M + H]+ = 1296.687), renin substrate ([M + H]+ =
1758.933), or ACTH clip 18–39 ([M + H]+ = 2465.199) (Ta-
ble 1).
monomers for discrete incorporation into oligomers. This
approach proved to be inefficient and, ultimately, syntheti-
cally intractable (4). We therefore decided to pursue the
post-synthetic modification strategy based on a methyl
orotate containing oligomer, thus exploring its feasibility as
a convertible nucleobase. Synthesis of the methyl orotate
monomer was achieved in five steps with an overall yield of
28%, compounds 2–6 were easy to handle and purified by
recrystallization or trituration.
The nucleobase submonomer was approached by the stan-
dard Fisher esterification of orotic acid 1, which gave methyl
orotate 2 in high yield. The methyl ester was chosen as a
simple, orthogonal protecting group to the Fmoc-protected
amine and the acid labile tert-butyl group for carboxyl pro-
tection. The alkylation of 2 with tert-butyl bromoacetate and
potassium carbonate gave 3 and acidolysis of the tert-butyl
ester gave (methyl orotate-1-yl)acetic acid (4, Scheme 1).
The alkylation at N1 of methyl orotate was confirmed by 2D
NMR (gHMBC) spectroscopy. Compound 3 showed cou-
pling from the methylene protons of CH2-COO-t-Bu (singlet
at δ 4.60) to both C6 (δ 143.8) and C2 (δ 151.0), while no
coupling to C4 (δ 162.2) was observed.
Binding analysis
Poly(riboadenylic acid) (poly(rA)) and poly(deoxyribo-
adenylic acid) (poly(dA)) were purchased from Sigma (Mil-
waukee, Wisconsin). Thermal denaturation of PNA:NA
complexes (~2 µmol/L in nucleobases) were measured at
260 nm with a temperature ramp of 0.5 °C/min. A minimum
of two independent samples were run in triplicate and the Tm
was estimated by the first derivative method. Identical ionic
conditions (150 mmol/L NaCl, 10 mmol/L Na2HPO4,
1 mmol/L EDTA at pH 7.0) were used for both the thermal
denaturation studies and the determination of binding
stoichiometry by the method of continuous variations. The
error associated with the reported Tm values is 0.5 °C.
Binding stoichiometry was determined by linear least-
squares fitting of the curves. For the example, the two lines:
y = 0.2935x + 0.4744 and y = 0.3028x + 0.064 fit the experi-
mental data and gives the binding stoichiometry (calcd. 0.66;
found: 0.69χPNA).
The synthesis of uracil-5-methylcarboxylate, as shown in
Scheme 2, began with 5-hydroxymethyluracil, which is eas-
ily synthesized in high yield by the well-known reaction of
uracil with formaldehyde (11). The oxidation of 5-
hydroxymethyluracil to the carboxylic acid may be achieved
in one step, but we found that 5-formyluracil cleanly precip-
Results and discussion
Synthesis of PNA monomers
Our initial approach to incorporate C6 derivatives of ura-
cil into PNA involved the synthesis of individual orotamide
© 2005 NRC Canada

Hudson and Wojciechowski
1737
Scheme 3. Reagents and conditions: (a) ClCH2CO2-t-Bu, DCM, 4 h at 0 °C, 12 h at ambient; (b) Fmoc-NHS, DIPEA, 0–20 °C, 4 h.
H
N
H
O
H
N
O
Cl
b, 61%
a, 76%
NH2
Fmoc
H2N
N
H
O
H2N
O
Scheme 4. Reagents and conditions: (a) DCC, HOBt, DMF, 24 h; (b) CF3COOH, DCM, Et3SiH.
O
O
O
R1
NH
R1
R1
6. 82%
14. 83%
NH
5. 55%
13. 70%
NH
R2
N
O
O
R2
N
O
R2
N
O
a.
b.
4 or 12
O
O
O
O
OH
+
N
Fmoc
N
Fmoc
H
O
N
H
OH
N
H
O
N
Fmoc
N
H
O
6 or 14
5 or 13
6. R1= CO2CH3 R2=H
14. R1= H R2=CO2CH3
5. R1= CO2CH3 R2=H
13. R1= H R2=CO2CH3
4. R1= CO2CH3 R2=H
12. R1= H R2=CO2CH3
itated during the course of the reaction using potassium per-
sulfate and catalytic silver nitrate by a modified procedure
of Ziegler and Brossmer (12). The 5-formyluracil may sub-
sequently be dissolved in boiling water and subjected to an-
other round of oxidation with potassium persulfate and
catalytic silver nitrate to give the carboxylic acid. Next
Fisher esterfication, alkylation, and acidolysis as previously
described gave (uracil-5-methylcarboxylate-1-yl)acetic acid,
compound 12. Although the C5-methyl carboxylate
nucleoside has been prepared by carbonylation of 5-iodo-2-
deoxyuridine (15), the stepwise process previously described
is preferable to us because it is technically very simple, may
be done on a large (multigram) scale in good chemical
yields, and furnishes intermediates that are useful for other
studies (4). Once the C5-ester had been installed, compound
10 was alkylated at N1 to give 11 and the regiochemistry for
this reaction was confirmed by 2D NMR (gHMBC) spec-
troscopy. Compound 11 showed coupling from the methy-
lene protons of CH2-COO-t-Bu (singlet at δ 4.43) to both C6
(δ 149.6) and C2 (δ 152.3), as well as CH(6) (singlet at δ
8.19) to the methylene carbon (δ 49.9).
Next, the Fmoc backbone was prepared by a modified
procedure of Thomson et al. (14) (Scheme 3). An excess of
ethylenediamine was alkylated with tert-butyl chloroacetate,
followed by aqueous work up. The product, tert-butyl N-(2-
aminoethyl) glycinate, was then reacted with Fmoc-NHS for
4 h, extracted and carefully acidified with 1 equiv. of HCl in
ether (5 mol/L) to give the Fmoc backbone HCl salt
(Scheme 3). In the condensation to the nucleobase-1-yl-
acetic acid, the Fmoc backbone HCl salt was neutralized by
extraction against sodium bicarbonate and used as the free
base.
methylcarboxylate monomers were used in the solid-phase
peptide synthesis of oligomers.
Synthesis of PNA oligomers
Incorporation of the methyl orotate (MO) monomer into
PNA hexamers was done using a Rink amide resin using
standard conditions for Fmoc-based oligomerization chemis-
try. Hexamers were synthesized containing thymine and ter-
minally, internally, or doubly substituted MO derivatives 8–
10. The methyl orotate monomer, and the subsequently in-
corporated methyl orotate unit, was found to be completely
compatible with the solid-phase synthesis; no saponification,
transesterification, or other loss of the methyl ester func-
tional group was found.
The amide linkage to the resin allowed for reaction of
resin bound oligomers 8–10 with hydroxide or an amine.
Standard acidolysis with TFA–Et3SiH released the oligomers
from the resin to give methyl orotate 8–10, orotic acid 11–
13, or orotamide 14–17 containing PNAs (Table. 2). Hydro-
lysis of resin-bound methyl orotate oligomers was performed
using NaOH in THF–H2O. The orotic acid oligomers were
released from the resin by acidolysis and RP-HPLC showed
one major peak with minimal byproducts. Aminolysis of
methyl orotate allows for the introduction of various substi-
tuted amides, and incorporation of 3-(N,N-dimethyl-
amino)propylamide or ethanolamide was accomplished by
treating the resin with neat amine at 55 °C for 6 h, and
cleavage form the resin was performed as described. For ex-
ample, resin-bound oligomer 10 was converted to 16 as pre-
viously described (Fig. 2A), isolated as the only major
product by RP-HPLC (Fig. 2B), and identified by mass
spectrometry (Fig. 2C).
Condensation of 4 and 12 to the neutralized Fmoc back-
bone was achieved with DCC–HOBt, and subsequent
acidolysis of the tert-butyl ester gave the target methyl
orotate monomer 6 and uracil-5-methylcarboxylate mono-
mer 14 (Scheme 4). Both the methyl orotate and uracil-5-
Synthesis of uracil-5-methylcarboxylate and uracil-5-car-
boxamide containing oligomers (19, 20) proceeded in a
similar fashion. The uracil-5-methylcarboxylate (UM)
was introduced into the internal position of a hexamer
Ac-TTTUMTTK-NH2 (19) and converted to the 3-(N,N-
© 2005 NRC Canada

1738
Can. J. Chem. Vol. 83, 2005
Fig. 2. (A) (CH3)2N(CH2)3NH2, 55 °C, 6 h. (B) RP-HPLC trace of crude oligomer 16. (C) MALDI-MS of purified 16 (calcd. mass
MH+: 2012.9076; observed MH+: 2012.7640).
(A)
O
N
O
N
O
N
O
N
O
N
O
N
NH
NH
NH
NH
NH
NH
NHBOC
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
PE
G
N
H
H
N
N
N
N
N
N
N
HN
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
a, b
O
N
N
O
N
O
N
O
N
O
N
O
N
O
N
NH
NH
NH
NH
NH
NH
NH2
NH2
HN
HN
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
HN
N
H
N
H
N
H
N
H
N
H
N
H
O
O
(B)
(C)
2012.7640
1.20
100
1.00
0.80
0.60
0.40
0.20
0.0
%
calibrant
2466.1624
0
1200
1600
2000
2400
2800
3200
3600
10
20
30
40
50
60
m/z
Minutes
Table 2. Summary of Tm values for triplex complexes formed between PNA hexamers 7–18 and poly(rA) or poly(dA).
Entry
PNA
Tm(RNA)
Tm(DNA)
∆Tm(RNA)
∆Tm(DNA)
7
Ac-TTTTTTK-NH2
62.0
47.2
22.5
28.0
46.0
18.3
23.5
56.5
34.0
—
57.0
35.8
25.5
17.0
35.0
24.3
—
—
—
8
Ac-MOTTTTTK-NH2
Ac-TTTMOTTK-NH2
Ac-MOTTMOTTK-NH2
Ac-OATTTTTK-NH2
Ac-TTTOATTK-NH2
Ac-OATTOATTK-NH2
Ac-OMTTTTTK-NH2
Ac-TTTTOMTTK-NH2
Ac-OMTTOMTTK-NH2
Ac-TTTOMeTTK-NH2
Ac-TTTCTTK-NH2
–14.8
–39.5
–34.0
–16.0
–43.7
–38.5
–3.5
–21.2
–31.5
–40.0
–22.0
–32.7
—
9
10
11
12
13
14
15
16
17
18
19
20
47.2
28.0
30.0
29.5
—
–9.8
–29.0
–27.0
–27.5
—
–28.0
—
33.5
19.0
59.0
70.0
–28.5
–43.0
–3.0
Ac-TTTUMTTK-NH2
Ac-TTTUATTK-NH2
—
—
—
+8.0
—
Notes: Abbreviations in use for modified nucleobases: methyl orotate (MO), orotic acid (OA), uracil-6-{N-[3-(N′,N′-dimethylamino]propyl}carboxamide
(OM), uracil-6-(N-2-hydroxyethyl)carboxamide (OMe), uracil-5-methylcarboxylate (UM), uracil-5-{N-[3-(N′,N′-dimethylamino]propyl}carboxamide (UA),
lysine (K), acetyl (Ac).
© 2005 NRC Canada

Hudson and Wojciechowski
1739
orotate (uncharged group) or orotic acid (negatively charged
group) suffered a much greater destabilization. The greatest
destabilization of complexes formed with both RNA or
DNA contained the orotic acid insert, thus implying that
electrostatic interactions are important. Single substitution
internal to the sequence (9, 12, 15, 17) and doubly substi-
tuted oligomers (terminal and internal: 10, 13, 16) had a
large detrimental effect. Such large effects for internal sub-
stitution suggest that the substituents exert a significant
steric effect. For comparison, the internal substituted orotic
acid hexamer 12 has a decrease in Tm that is comparable to
the oligomers containing a mismatch: (Ac-TTTCTTK-NH2,
18, ∆Tm = −43 °C). Even the putative beneficial effect of a
pendant positive charge (15, 16) is insufficient to recover the
binding affinity of the hexamers.
Fig. 3. Job plot of poly(rA) and the orotamide hexamer 15 (sin-
gle internal substitution) in 150 mmol/L NaCl, 10 mmol/L
Na2HPO4, and 1 mmol/L EDTA at pH 7.0 buffer.
0.5
0.45
0.4
0.35
~ 2:1
0.3
In stark contrast, our preliminary results show that the
PNA containing an internal uracil-5-carboxamide stabilizes
triple-helix formation with complementary RNA (Table 2,
20). This result parallels those found in the chemistry of
DNA and RNA, but in PNA the stabilization is larger in
magnitude. The introduction of a single internal C5
carboxamide with a pendant positively charged group into
an oligodeoxynucleotide has been shown to have a very
modest effect on thermal stability (at 100 mmol/L NaCl) for
duplexes formed with complementary DNA or RNA (18,
19), although larger effects were observed under low-salt
conditions (20). The stabilization in these cases was attrib-
uted to increased base stacking through a five-membered
ring formed by H bonding of the amide hydrogen with the
C4 carbonyl of uracil and (or) by the pendant cationic
charge (19). Other studies with oligonucleotides capable of
forming triple helices demonstrated a destabilization of the
complex when the polypyrimidine oligonucleotide possessed
a single internal C5-carboxamide-modified uracil (18, 21).
Granted, these are different sequence contexts and different
structural backbones, thus comparisons between PNA and
NA systems need to be made cautiously. In addition, for the
triplex system described herein, each PNA strand carries a
modification, so that each complex has a total of two modi-
fied residues; whereas the studies referenced on duplexes
possess a single modified residue. However, it is noteworthy
that the introduction of a single internal modification bear-
ing a pendant positively charged group into the PNA strand
is more stabilizing to the triplex formed than incorporation
of a backbone modification based on lysine (22), but not as
significant as a positively charged nucleobase analog that
makes a specific H-bonding interaction with the complemen-
tary base — the so-called G-clamp (23). Such pendant posi-
tive charges incorporated into PNA via the nucleobases may
benefit the binding affinity, sequence discrimination (24),
accelerate hybridization and strand invasion (25–27), and fa-
vourably affect the intracellular accumulation (28) for in
vivo applications.
0.25
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mol fraction of PNA 15
dimethylamino)propylamide
TTTUATTK-NH2 (20).
giving
oligomer
Ac-
Hybridization studies of PNA containing C5 and C6
derivatives of uracil
The hybridization of methyl orotate, orotic acid, and
orotamide substituted hexamers 8–17 and C5-substituted
ester and amide 19 and 20 was studied by temperature-
dependent UV absorbance. The binding stoichiometry for
the modified oligomers containing C5 and C6 carboxamides
with poly(rA) was determined by the method of continuous
variation, commonly referred to as Job plots (15). This anal-
ysis revealed a 2:1 ratio, which is consistent with triplex for-
mation (Fig. 3, shown for orotamide). Presumably, this
triplex is composed of an antiparallel Watson–Crick (16)
binding domain and a parallel Hoogsteen (17) binding do-
main as seen for covalently linked bisPNAs (10). The use of
unlinked binding domains is useful in this case because an
antiparallel orientation between the two domains is not en-
forced and the oligomers are free to adopt the most favour-
able binding arrangement. If the C6 derivatives had been
found to benefit triplex formation, elucidation of the strand
orientation would be more relevant (vide infra).
The thermal stability (Tm) of triplex formation was evalu-
ated near physiological ionic conditions (150 mmol/L NaCl,
10 mmol/L Na2HPO4, 1 mmol/L EDTA at pH 7.0). The
hexamers were hybridized to poly(rA) or poly(dA) in a 2:1
ratio at a concentration of ~2 umol/L per nucleobase. The
compounds were all well-behaved and showed a monophasic
hyperchromic shift. The introduction of C6-modified resi-
dues into the hexamers (8–17) caused a drop in Tm in all
cases as compared to an unmodified PNA control sequence
(Ac-TTTTTTK-NH2, 7), as summarized in Table 2. Single
modified residues located at the N terminus of the sequence
(8, 11, 14) were better tolerated than at an internal position
(9, 12, 15). The N-terminal modifications show a trend that
can be rationalized predominately on electrostatic interac-
tions. For instance, the introduction of an orotamide, with a
pendant positively charged group, at the N terminus of the
hexamers had only a weakly destabilizing effect. Complexes
formed with hexamers containing an N-terminal methyl
In summary, we have described the synthesis of a methyl
orotate and uracil-5-methylcarboxylate monomers that are
effective convertible nucleobases, compatible with an on-
resin conversion strategy. Incorporation of the esters into oli-
gomers allows for aminolysis in high yield and purity. How-
ever the C6-modified PNA2:NA triple helices do not tolerate
this substitution, while the C5-carboxamide substitution sta-
bilized triplex formation. We are extending these investiga-
© 2005 NRC Canada

1740
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Acknowledgements
The authors thank the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Academic
Development Fund at the University of Western Ontario for
support of this work. Professor G. Lajoie, Dr. Amanda
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© 2005 NRC Canada

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