ABSTRACT
Adenine, thymine and cytosine PNA monomers have been prepared using 3-amino-1,2-propanediol as a starting material. The benzoyl group was used
to protect the exocyclic amines of the heterocyclic bases of A and C PNA
monomers and the backbone primary amine was protected with the
monomethoxytrityl group. The thymine and cytosine PNA monomers were used in
conjunction with standard DNA synthesis monomers to produce chimeric PNA DNA
(PDC) oligomers. Ultraviolet melting studies confirmed that these oligomers
form stable hybrids with complementary DNA strands and that mismatches in the
DNA but more so in the PNA sections lead to duplex destabilisation.
Since the first report of the synthesis of peptide nucleic acids (PNAs) (
1
) there have been many reports describing the binding of PNA to DNA and to RNA (
2
), the biological applications of PNA (
3
-
6
) and structural variations of the basic PNA structure (Fig.
1
) including the use of elongated (
7
,
8
) and chirally modified backbones (
9
,
10
). Here we describe the synthesis of PNA monomers which are compatible with
solid-phase DNA synthesis and the assembly on solid-phase of DNA-PNA chimeras (Fig.
2
) which have potential applications in biology.
PNAs are uncharged at physiological pH and one of the major drawbacks to their
use is their poor water solubility. However the lack of backbone charge also
gives rise to unusual hybridisation behaviour which is potentially of great
value in molecular biology. There are no electrostatic interstrand repulsions
when PNAs hybridise with nucleic acids and this leads to higher thermodynamic
stability than structurally analogous DNA-DNA or RNA-DNA hybrids (
11
). In addition, in contrast to DNA-DNA hybrids, PNA-DNA hybrids are stable under low salt conditions and it has been
shown that PNA and DNA strands have a faster rate of binding relative to DNA-DNA strands (
12
).
It is possible that PNA-DNA chimeric oligomers in which both types of monomeric unit are present
in a single chain might combine the favourable hybridisation characteristics of
PNA with the high water solubility of DNA. Moreover, the structure of such
molecules, if it bears a resemblance to DNA, might be compatible with some
enzyme catalysed reactions. Such compounds could therefore be of use in PCR,
DNA sequencing, antisense inhibition studies and other technologies.
Our strategy to produce such molecules was to assemble a DNA chain on a solid
support using standard phosphoramidite coupling chemistry and then to couple
the PNA monomers in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Fig.
2
). In order to achieve this objective we prepared PNA monomers with the same
protection chemistry as conventional DNA phosphoramidites. From the outset it
was clear that
t
-butyloxycarbonyl (boc) protection of the PNA N-terminus was not desirable since the TFA used in boc-deprotection is not compatible with oligodeoxynucleotides
which depurinate rapidly in acidic media. 9-Fluorenylmethoxycarbonyl (Fmoc) protection was equally unsuitable because
its removal with base would lead to cleavage of the oligonucleotide from the
solid support. Bergmann
et al
. have shown that these problems can be overcome (
13
) but in the interests of simplicity and convenience we opted to use the
monomethoxytrityl group for N-terminus protection. We adopted a similar approach to that used by Will
et al
. in PNA synthesis (
14
)
and by van der Laan
et al.
(
15
)
and Stetsenko
et al.
(
16
)
in the synthesis of oligo dT and U/dT PNA-DNA hybrids. Benzyloxycarbonyl protection of the exocyclic amino groups
of the A and C monomers has been used extensively in PNA synthesis but is not
suitable for PNA-DNA chimeras since the hydrogenolysis or acidolysis required to remove
this group would add a further deprotection step which could damage the DNA
portion of the chimera. Therefore the PNA bases were protected with the benzoyl
group, the same as the bases of the corresponding DNA phosphoramidites.
The PDC oligomers were assembled with the DNA section at the 3'-end and the PNA section at the 5'-end (N-terminus, Fig.
2
). In principle this would allow for template directed extension of the DNA
chain in subsequent enzymic reactions. Previously a modified PNA thymine
monomer bearing a dimethoxytrityloxyethyl group has been used to couple PNA
thymine units to the `3'-end' of DNA (
22
)
(the opposite orientation). In our work DNA sections were synthesised using standard phosphoramidite chemistry and the
modified nucleoside 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite (
23
) was added as a linker between PNA and the 5'-end of the DNA chain. The PNA monomers were added using TopPipU as
activating agent and deprotection of the monomethoxytrityl terminal amino functions prior to each PNA coupling step
was carried out using TCA (3% w/v in dichloromethane). Capping of unreacted
amino groups was achieved using acetic anhydride in pyridine. Solution and
solid-phase reactions showed that the adenine PNA monomer failed to couple
efficiently under these conditions and this is the subject of continuing work.
In the present study only cytosine and thymine PNA monomers were used for
oligomer synthesis. The chimeric oligomers were cleaved from the solid support
and the exocyclic amino protecting groups were removed by heating the resin in
concentrated aqueous ammonia at 55oC for 6 h. The crude products were purified by reversed-phase HPLC, desalted by Sephadex gel filtration and analysed by
capillary electrophoresis (Figs
3
and
4
). This single purification protocol gave oligomers with only minor levels of
impurities.
Ultraviolet melting studies (Table
1
;
24
) of the PDC sequences Ac-tttcttTGCCAT-3' and Ac-ttttttTGCCAT-3' (PDC = PNA-DNA chimera, Ac = acetyl on the
amino group of the terminal PNA residue, DNA residues in upper case, PNA
residues in lower case) with their DNA complements indicate increased stability
of the PDC-DNA duplex with respect to the native DNA duplexes (an increase in
T
m
of 10.2oC in the former and 7.7oC in the latter case). The inter-strand interaction between the chimera and the complementary DNA
strand was shown to occur in a sequence specific manner. In the case of Ac-ttttttTGCCAT-3' a G[middot]t mismatch in the centre of the PNA part of the
chimeric strand opposite the DNA strand gave a drop in
T
m
of 16.2oC whereas a C[middot]C mismatch in the DNA part of the chimera gave a drop in
T
m
of only 2.8oC. This suggests that the duplex is stabilised much more by PNA-DNA interactions than DNA-DNA interactions and that mismatches in the PNA section are
very destabilising. This was confirmed with the sequence Ac-tttcttTGCCAT-3' in which a PNA-DNA A.c mismatch caused a massive drop in
T
m
of 37.3oC whereas a C[middot]C mismatch involving the DNA portion of the chimera caused a drop
in
T
m
of only 1.9oC.
We have demonstrated that PNA-DNA chimeric oligomers can be synthesised in a stepwise fashion using PNA
monomers which are compatible with DNA synthesis and we have confirmed that the
chimeras hybridise specifically to complementary DNA. The PDC oligomers form
more stable complexes with complementary DNA than do the equivalent DNA
sequences.
All solvents were of analytical grade; dichloromethane (DCM),
diethylcyclohexylamine (DECHA) and pyridine were distilled over CaH
2
; methanol (MeOH) was distilled over Mg and I
2
; tetrahydrofuran (THF) was distilled over sodium and benzophenone,
N
,
N
-dimethylformamide (DMF) was of peptide synthesis grade and DNA synthesis
grade acetonitrile was purchased from Perkin-Elmer Ltd. TopPipU was obtained from Novabiochem. All other chemicals were
supplied by Aldrich, Sigma or Fluka.
Table 1
1
H and
13
C NMR were recorded on Brucker 250AC and Brucker 200WP spectrometers. Positive
ion Fast Atom Bombardment (FAB) mass spectra were recorded on a Kratos MS50TC
spectrometer using a thioglycerol matrix, chemical ionisation (C.I.) mass
spectra were recorded on a VG analytical 70-250 SE Normal Geometry Double Focus Mass Spectrometer and electrospray
mass spectra were recorded on a VG Biotech Platform.
Flash chromatography was carried out using silica gel 60 (Fluka). Thin layer
chromatography (TLC) was carried out on aluminium sheets, silica 60 F
254
, 0.2 mm layer (Merck) using the following solvent systems; A =
n
-butyl alcohol/acetic acid/H
2
O (3:1:1 v:v), B = DCM/ethyl acetate (1:1 v/v), C = DCM/MeOH (9/1 v/v). Products
were visualised using ninhydrin (1% w/v in EtOH) with heating for 5 min, UV
irradiation at 254 nm, iodine oxidation or phosphomolybdic acid (10% w/v in
water) with heating.
To an ice-cooled solution of racemic 3-amino-1,2-propanediol (10 g, 0.11 mol) in water (250 ml) was added
boc anhydride (25 g, 0.12 mol). The mixture was allowed to warm to room
temperature and the pH was maintained at 10.5 by the addition of aqueous NaOH
(2N). The solution was concentrated to a paste which was triturated with DCM
(500 ml). The suspension was filtered and the organic phase was dried (MgSO
4
) then evaporated to give the diol
2
(20.4 g, 97%) as an oil which solidified upon freezing.
R
f
0.75 (A), 0.60 (B), 0.70 (C). [delta]
H
(CDCl
3
); 1.39 (s, 9H, (C
To diol
2
(5 g, 26.1 mmol) in water (50 ml) was added NaIO
4
(6.8 g, 1.2 eq.) at room temperature with stirring. After 3 h the mixture was
filtered, extracted with DCM (5 * 100 ml) and the combined organic phases were dried (MgSO
4
) and concentrated to yield the aldehyde
3
(3.9 g, 94%) as an oil.
R
f
0.70 (A), 0.15 (B), 0.50 (C); [delta]
H
(CDCl
3
); 1.38 (s, 9H, (C
To
t
-butoxycarbonylaminoacetaldehyde
3
(5.13 g, 32.2 mmol) in MeOH (100 ml) was added ethyl glycinate hydrochloride
(11.2 g, 2.5 eq.) and NaBH
3
CN (2.02 g, 1 eq.) with stirring at room temperature. After 16 h the mixture was
evaporated, the residue dissolved in water (100 ml) and the pH adjusted to 8.0 by the addition of
aqueous NaOH (2 M). The solution was extracted with DCM (5 * 150 ml) and the combined organic fractions were dried (MgSO
4
) and evaporated to give
4
as a colourless oil (3.42 g, 43%) after flash silica column chromatography (DCM
+ 0-4% MeOH).
R
f
0.55 (A), 0.05 (B), 0.15 (C), [delta]
H
(CDCl
3
); 1.22 (t, 3H, J = 7.1 Hz, CH
2
C
To a solution of thymine (10.0 g, 79.3 mmol) and KOH (17.1 g, 3.8 eq.) in water
(50 ml) was added dropwise, with stirring, bromoacetic acid (16.5 g, 1.5 eq.) in water (25 ml) at 40oC over 30 min. After 2 h the solution was cooled to room temperature and
the pH was adjusted to 5.5 by the addition of conc. HCl. A precipitate was
removed after 2 h at -4oC and the pH of the filtrate was adjusted to 2.0 (conc. HCl). The
acid
5
(12.5 g, 86%) was collected and dried over P
2
O
5
.
R
f
0.10 (A), [delta]
H
(d
6
-DMSO) 1.74 (s, 3H, Thy-C
Cytosine (10.0 g, 0.09 mol) was suspended in pyridine (750 ml) and benzoyl
chloride (21.0 ml, 2 eq.) added dropwise with stirring over 3 h. The reaction
mixture was stirred at room temperature for a further 1 h after which the pH
was adjusted to 5.0 by the addition of 4 N HCl. The resulting suspension was
stirred at room temperature for a further 2 h and the precipitate collected,
washed with EtOH (3 * 100 ml) and dried
in vacuo
over P
2
O
5
to give the title amide (12.0 g, 62%). The product was only sparingly soluble
in organic solvents and water and therefore no NMR data were obtained. m/z
(f.a.b.) = 216.0778 (M
+
+ H) [C
11
H
10
N
3
O
2
requires 216.0773].
N
(4)-benzoylcytosine
6
(5.00 g, 23 mmol) and potassium carbonate (3.20 g, 1 eq.) were suspended in DMF
(100 ml) and methyl bromoacetate (2.24 ml, 1 eq.) was added dropwise with
stirring over 3 h. Stirring was continued at room temperature for 16 h then the
suspension filtered. The filtrate was evaporated to dryness, water (75 ml) and
HCl (4N, 2.5 ml) added and the precipitate collected and suspended in water (40
ml). NaOH (2 N, 30 ml) was added and the solution was stirred at room
temperature for 30 min then filtered. To the filtrate was added conc. HCl (5
ml), and the precipitate was collected and dried
in-vacuo
to afford the title acid (2.80 g, 45%).
R
f
0.13 (A), 0.05 (C); [delta]
H
(d
6
-DMSO) 4.66 (s, 2H, NC
A 60% dispersion of sodium hydride in paraffin, (3.26 g, 0.82 mol) was washed
with hexane (3 * 100 ml) then suspended in DMF (250 ml) at 0oC. To this was added adenine (10.0 g, 0.74 mol) slowly with
stirring. On completion of addition the reaction mixture was warmed to room
temperature and stirred for 2 h before methyl bromoacetate (12.9 ml, 1.8 eq.)
was added over 3 h with continual stirring. The reaction mixture was stirred at
room temperature for a further 16 h then the solution was concentrated to a
paste. Water (100 ml) was added and the precipitate collected and
recrystallised from EtOH/water (3:2 v/v, 120 ml), to give the title ester (11.4
g, 75%).
R
f
0.55 (A), 0.12 (C). [delta]
H
(d
6
-DMSO) 3.68 (s, 3H, CO
2
C
Compound
9
(5.00 g, 0.024 mol) was coevaporated with pyridine (3 * 10 ml) then redissolved in pyridine (50 ml). Benzoyl chloride (3.1 ml,
1.1 eq.) in pyridine (50 ml) was added dropwise over 3 h at room temperature
and stirring was continued for 16 h. Water (100 ml) was added then the reaction
mixture was extracted with DCM (3 * 100 ml). The combined extracts were washed with NaHCO
3
(0.5N, 3 * 100 ml), NaCl (sat., 100 ml), dried (Na
2
SO
4
), then evaporated to an oily residue. Purification by flash column
chromatography [SiO
2,
DCM/MeOH (0-6%)] yielded
10
(4.71 g, 64%) as a white solid.
R
f
0.61 (A), 0.10 (B), 0.25 (C). [delta]
H
(CDCl
3
); 3.76 (s, 3H, CO
2
C
Compound
10
(4.41 g, 0.14 mol) was suspended in MeOH (80 ml) at 0oC. NaOH (2N, 80 ml) was added and the reaction mixture stirred at 0oC for 30 min. The solution was washed with DCM (2 * 80 ml) then the pH adjusted to 1 by the addition of conc. HCl
(5 ml). The title acid (3.43 g, 72%) was collected and dried over P
2
O
5
under vacuum.
R
f
0.35(A), 0.1(C); [delta]
H
(d6 DMSO); 5.27 (s, 2H, C
Ethyl-
N
-[2-(
t
-butoxycarbonylamino)ethyl] glycinate
4
(2.75 g, 11 mmol) and thymine-
N
(1)-acetic acid
5
(2.05 g, 1 eq.) were dissolved in DMF (25 ml) and triethylamine (4.42 ml, 3
eq.) was added. TopPipU (4.2 g, 1.0 eq.) was added and the reaction mixture was
stirred at ambient temperature for 16 h. The solvent was removed by evaporation
and DCM (250 ml) was added to the residue. The resultant solution was washed
with NaHCO
3
(0.5 N, 3 * 100 ml), citric acid (10% w/v, 2 * 100 ml), and saturated NaCl (100 ml), dried (MgSO
4
) then concentrated to an oil which was subjected to flash column chromatography
[DCM/MeOH (0-3%)]. The ester
12
(3.2 g, 71%) was isolated as a foam.
R
f
0.75 (A), 0.15 (B), 0.33 (C); [delta]
H
(CDCl
3
); (two rotational isomers in the ratio 2:1 were detected due to restricted
rotation about the tertiary amide bond) 1.25 (t, 3H, J = 7.2 Hz, C
Ethyl-
N
-[2-(
t
-butoxycarbonylamino)ethyl] glycinate
4
and
N
(4)- benzoyl cytosine-
N
(1)-acetic acid
8
were condensed as previously described for amide
12
. The title compound (74%) was isolated as a pale pink foam following flash
column chromatography [SiO
2
, DCM/MeOH (0-3%)];
R
f
0.33 (B), 0.55 (C); [delta]
H
(CDCl
3
); (two rotational isomers were observed) 1.22 (t, 3H, J = 7.2 Hz, CH
2
C
Ethyl-
N
-[2-(
t
-butoxycarbonylamino)ethyl] glycinate
4
and
N
(6)- benzoyladenine-
N
(9)-acetic acid
11
were condensed as previously described for amide
12
. The title compound (78%) was isolated as a white solid following flash column
chromatography [SiO
2
, DCM/MeOH (0-6%)].
R
f
0.85 (A), 0.10 (B), 0.35 (C); [delta]
H
(CDCl
3
); 1.18 (t, 3H, J = 7.2 Hz, CH
2
C
Boc protected amine
12
(2.00 g, 4.85 mmol) was treated with trifluoroacetic acid (10 ml). After 30 min
the reaction mixture was evaporated to dryness
in vacuo
,
coevaporated with toluene (5 * 10 ml) followed by pyridine (2 * 10 ml) then resuspended in a further volume of pyridine (20 ml).
4'-Methoxytrityl chloride (1.65 g, 1.1 eq.) and 4-dimethylaminopyridine (DMAP) (25 mg) were added and the
mixture stirred at ambient temperature for 16 h. Water (30 ml) was added and
the mixture was extracted with DCM (3 * 70 ml). The combined organic phases were dried (MgSO
4
), then evaporated to an oil. The title amine (2.55 g, 90%) was isolated as a
white foam following flash column chromatography [SiO
2
, DCM with MeOH (0-3%)] using silica which had been pre-equilibrated with DCM containing triethylamine (1% v/v) to prevent
detritylation.
R
f
0.20 (A); [delta]
H
(CDCl
3
); (as with
12
, two rotational isomers were detected) 1.23 (t, 3H, J = 7.2 Hz, C
Boc protected amine
13
was converted to the corresponding monomethoxytrityl protected amine as
described for amine
12
. Following flash column chromatography [(SiO
2
, DCM/MeOH (0-4%)] the title amine (81%) was isolated as a colourless foam.
R
f
0.22 (B), 0.35 (C); [delta]
H
(CDCl
3
); (two rotational isomers were detected) 1.16 (t, 3H, J = 7.1 Hz, C
Boc protected amine
14
was converted to the corresponding monomethoxytrityl protected amine as
described for amine
12
. Following flash column chromatography [(SiO
2
, DCM/MeOH (0-2%)] the title amine (83%) was isolated as a colourless foam
R
f
0.10 (B), 0.25 (C); [delta]
H
(CDCl
3
); (two rotational isomers were detected) 1.17 (t, 3H, J = 7.1 Hz, C
The ester
15
(1.34 g, 2.3 mmol) was dissolved in MeOH (60 ml) and NaOH (2 N, 40 ml) added.
The mixture was stirred at room temperature for 2 h. then DOWEX (pyridinium
form) was added until neutral (wet pH paper). The suspension was filtered and
the resin washed with MeOH (3 * 50 ml). The filtrate was evaporated to dryness
to yield acid
18
as a white solid (1.40 g, 96%).
R
f
0.05 (B), 0 (C); [delta]
H
(d6 DMSO); (two rotational isomers were detected) 1.74 (s, 3H, Thy-C
Ester
16
was hydrolysed as described for ester
15
. The title acid (70%) was obtained as a white solid
R
f
0.05 (C); [delta]
H
(d6 DMSO) (two rotational isomers were detected) 2.23 (m, unresolved, 2H,
MMTrHNC
Ester
17
was hydrolysed as described for ester
15
. The title acid (97%) was obtained as a white solid
R
f
0.00 (B), 0.15 (C); [delta]
H
(d6 DMSO); (two rotational isomers were detected) 2.18 (m, unresolved, 2H,
MMTrHNC
The DNA sections were synthesised on an ABI 394 DNA/RNA synthesiser on the 1.0 [mu]mol scale using standard phosphoramidite chemistry. 5'-(4-Methoxytrityl)-amino-5-'deoxythymidine phosphoramidite (
19
) was added as the final nucleoside to act as a linker between PNA and DNA. The
resin was removed from the DNA synthesis column and transferred to a glass
sinter funnel used for PNA synthesis. The PNA monomers were added manually with
agitation of the resin during each synthetic step by means of a stream of
nitrogen. PNA monomer coupling was carried out in DMF/pyridine solution (1:1
v/v, 0.1 M) using diethylcyclohexylamine as base and TopPipU (
20
) as activating group for the acid with a coupling time of 30 min. The
in situ
neutralisation approach was used to inhibit aggregation of the PNA chains and
to prevent elimination reactions caused by intramolecular cyclisations of the
PNA backbone. Deprotection of the monomethoxytrityl terminal amino function was
carried out using TCA (3% w/v, 3 * 2 ml, 10 min) followed by thorough washing with DCM (5 * 1 ml). The resin was filtered then washed with MeOH (5 * 1 ml) then DMF (5 *1 ml).
Capping of unreacted amino groups was achieved using acetic anhydride/pyridine
solution (10% v/v, 1 ml) for 5 min. The resin was washed with DMF (5 * 1 ml) before the cycle was started again as required.
The oligomer was cleaved from the solid support and the exocyclic amino
protecting groups removed by heating the resin in concentrated aqueous ammonia
(2 ml, 55oC) for 6 h.
The chimeric PDC oligomers were purified by reverse phase HPLC using: A = NH
4
OAc (0.1 M, pH 7.0), B = MeCN (22.5% v/v)/NH
4
OAc (0.1 M, pH 7.0) employing the following gradient: B = 0% 3.5 min, B = 10%
4.5 min, B = 100% 30 min, B = 0% 38 min at a flow rate of 3 ml/min. After
evaporation salts were removed by Sephadex gel filtration.
Chimeric oligomers synthesised.
PDC Ac-tttcttTGCCAT-3', Electrospray mass spec. M 3388.53 C
125
H
161
N
46
O
58
P
5
requires 3390.80 (average mass), 3389.00 (monoisotopic mass). PDC ttttttTGCCAT-3', Electrospray mass spec. M 3405.43, C
126
H
162
N
45
O
59
P
5
requires 3405.81 (average mass), 3403.97 (monoisotopic mass).
Ultraviolet melting temperatures (
T
m
) were determined at 260 nm using a Perkin-Elmer Lambda 15 UV spectrometer equipped with a Peltier block and
controlled by an IBM PS2 computer. A heating rate of 0.5 K/min was used
throughout and the crude data was processed using the PECSS2 software package.
The oligonucleotides were dissolved in a buffer consisting of 0.14 M NaCl and
10 mM HEPES adjusted to pH 7.0. All experiments were repeated until three
values within 0.5 K of each other were obtained. Molar extinction coefficients
used for PNA and DNA bases were as follows: A, 15.4; T, 8.8; G, 11.7; C, 7.3 [mu]mol.cm.
T
m
values were determined from the maximum of the first derivative of the plot of
A
260
versus temperature.
See supplementary material available in NAR Online.
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