Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine

Article abstract of DOI:10.1039/c1ob06582c

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT_m ≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT _m ≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.

Full text of DOI:10.1039/c1ob06582c

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PAPER
Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of
2,6-diaminopurine, 2-aminopurine and thymine†
Andre´ H. St. Amant and Robert H. E. Hudson*
Received 15th September 2011, Accepted 27th October 2011
DOI: 10.1039/c1ob06582c
A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been
developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be
used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into
a PNA and was found to selectively bind to T (DTm ≥ +6 ◦C) in a complementary DNA strand. The
2AP monomer showed excellent discrimination of T (DTm ≥ +12 ◦C) over the other nucleobases. 2AP
also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching
dependent on the opposite base.
Introduction
Peptide nucleic acid (PNA) is a nucleic acid analogue containing
an N-(2-aminoethyl)glycine backbone.1 It has the ability to
form complementary base-pairing and has been investigated for
diagnostic and therapeutic properties.2 The automated synthesis of
PNA began with the Boc/Cbz‡ protecting group strategy,3 which
was superseded by Fmoc/Bhoc‡ for the milder conditions and
the lipophilic handle that the terminal Fmoc provides.4 Recently,
a Fmoc/Boc strategy has been investigated by our laboratory,5
which provides several useful features such as: additional levels
can also be paired with a complementary sequence containing
of orthogonality as the Bhoc protecting group is sensitive to
Fig. 1 Structure of the nucleobases used in this study: T (thymine), DAP
(2,6-diaminopurine), and 2AP (2-aminopurine).
thus increasing the stability of the duplex.7 The nucleobase
thiouracil to form duplex-invading PNAs.8 Despite its usefulness,
hydrogenation while Boc is not; the per-Boc nucleobases have
this unusual PNA is not commercially available, and this deficiency
excellent solubility in a range of organic solvents; decreased self-
has motivated us to develop a straightforward synthesis of the
aggregation due to a lack of H-bond donors; and compatibility
protected monomer.
The nucleobase 2-aminopurine (2AP) is an adenine analogue
with Fmoc/Bhoc monomers. The synthesis of guanine, adenine,
and cytosine Fmoc/Boc protected PNA monomers has been
reported,5 but the synthesis of the corresponding protected
thymine (T) was not undertaken at that time. Traditionally thymine
has been left unprotected in PNA synthesis, but there are reports
of low coupling for poly-T tracts that has been ascribed to on-resin
aggregation.6 As an alternative to the allyl ether-based protection
of thymine, that has already been demonstrated,6 we have extended
the Boc protecting group strategy, which is expected to increase the
organic solubility of growing poly-T tracts and reduce truncation
products caused by self-aggregation.
(Fig. 1) that possesses environmentally sensitive fluorescence
that has been exploited in the study of nucleic acid structures
and dynamics. The synthesis of an Fmoc-protected 2AP PNA
monomer with an unprotected exocyclic amine has been reported,9
but the detailed experimental conditions were not provided. The
binding-dependent fluorescence of 2AP in PNA is of interest to
our group and hence spurred the development of the synthesis of
an Fmoc/Boc protected PNA monomer.
2,6-Diaminopurine (DAP) is an adenine analogue (Fig. 1) that
is used for its ability to form three hydrogen bonds to thymine,
Results and discussion
Synthesis of protected PNA monomers
Department of Chemistry, University of Western Ontario, 1151 Richmond
St., London, Canada. E-mail: rhhudson@uwo.ca; Fax: +1-519-661-3022;
Tel: +1-519-661-2166
N-Boc-thymin-1-yl acetic acid (2) was prepared through the N-
Boc-protection of benzyl thyminyl-1-acetate10 (Scheme 1) which
furnished 1 in 95% yield under conditions similar to those previ-
ously reported by us for the Boc-protection of other nucleobases.5
Subsequent hydrogenolysis of the benzyl ester using Pd/C pro-
duced the acid 2 in quantitative yield. The hydrogenation of the
† Electronic supplementary information (ESI) available: 1H and 13C-NMR
spectra for compounds 1–14 and mass spec for PNAs, melt curves, and
fluorescence studies. See DOI: 10.1039/c1ob06582c
‡ Abbreviations: Boc = tert-butyloxycarbonyl; Cbz = benzyloxycarbonyl;
Fmoc = fluorenyloxycarbonyl; Bhoc = benzhydryloxycarbonyl.
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Scheme 2 Reagents and Conditions: (vi) EDC, HOBt, DCM, 0 ◦C to rt;
(vii) H2, Pd/C, acetone and/or MeOH, rt.
di(Boc)-2-aminopurin-9-yl acetic acid (12). Firstly, it was desirable
to work with a 6-chloro-2-aminopurine derivative to ensure
alkylation at the N9 position; however, conditions for successful
reductive dehalogenation were elusive. Both the benzyl and ethyl
esters of 6-chloro-2-aminopurin-9-yl acetate were synthesized,
but efficient hydrogenolysis of the chloro substituent was not
possible. Use of a Parr apparatus for higher H2 pressure (50 psi),
stoichiometric amounts of Pd/C, and a variety of solvents were
examined, but in all cases the 6-chloro group remained intact.
Attempts at photochemical dechlorination (xenon lamp, 1 to 24 h)
in THF only produced an intractable mixture. A substitution
reaction using thiourea to produce the 6-thio derivative was high-
R
yielding, but RANEYꢀ Ni desulfurization was only effective in
cases where the reactant was an ester and the nucleobase was
unprotected. Subsequent installation of the Boc groups, on the
free acid or esters of 2-aminopurin-9-yl acetate proved to be low-
yielding.
The most successful pathway to acid 12 was from the known
di(Boc)-2-amino-6-chloropurine,13 which was alkylated at the
N9 position with benzyl bromoacetate to afford 11a in 74%
yield, with the major impurity the N7 alkylated isomer. The
ester 11a was subjected to a simultaneous benzyl ester and
6-chloro hydrogenolysis with Pd/C in refluxing acetone using
HCOOH/Et3N as a hydrogen transfer agent.14 The ester was
observed to be cleaved much more quickly than the 6-chloro group,
producing intermediate 11b, which can be followed by HPLC. The
reaction was continued until the complete disappearance of 11b,
thus yielding 12 in 81% yield (Scheme 1). The acid 12 was coupled
with the Fmoc/Bn PNA backbone using EDC/HOBt to form
the monomer benzyl ester 13 (58%), which was then hydrogenated
using Pd/C to yield the PNA monomer 14 (95%, Scheme 2).
Scheme 1 Reagents and Conditions: (i) Boc2O, DMAP, THF, rt, 18 h; (ii)
H2, Pd/C, acetone and/or MeOH, rt, 2 h; (iii) NaHCO3, MeOH, 50 ◦C
1 h then rt, 18 h; (iv) benzyl bromoacetate, K2CO3, DMF, rt, 18 h; (v)
HCOOH, Et3N, Pd/C, acetone, reflux, 6 h.
pyrimidine ring for cytosine11 and the bis-Boc protected cytosine
has been noted, but was not observed in this instance for the N-
protected thymine.
The coupling of 2 with the Fmoc/Bn PNA backbone using
EDC/HOBt conditions produced 3 in 77% yield (Scheme 2).
The monomer benzyl ester was hydrogenated to yield the PNA
monomer 4 in 95% yield, again with no evidence of pyrimidine
hydrogenation.
The commercially available 2,6-diaminopurine was penta-Boc
protected, under conditions previously established for adenine,
to produce 5 in 96% yield. A mono-Boc deprotection12 was
performed on the N9 position of 5 using NaHCO3 in MeOH
to furnish 6 in 68% yield. The N9 position of 6 was alkylated
with benzyl bromoacetate to yield the benzyl ester 7 (97%), which
was hydrogenated to acid 8 in high yield (99%) using Pd/C. The
coupling of 8 with the Fmoc/Bn PNA backbone was performed
in the same manner as the thymine analogue, giving the benzyl
ester 9 (71%), which, after hydrogenation, yielded PNA monomer
10 in 96% yield (Scheme 2).
PNA synthesis
PNAs were synthesized on an Applied Biosystems 433A peptide
synthesizer using FastMoc chemistry. The syntheses were per-
formed on a 5 mmol scale using 5 equivalents of the monomers.
The peptides were cleaved from the resin and isolated/purified
according to literature procedures.15
The coupling performance of the thymine monomer 4 was in-
distinguishable from the commercially available T PNA monomer.
Successful peptide syntheses have been performed using a mixture
of 4 and commercial T monomer with no deleterious conse-
quences. A peptide with a penta(T) tract has been synthesized
using 4 with no evidence of premature truncation of the sequence
The synthesis of the 2AP monomer presented much more of
a challenge. We explored a variety of synthetic routes to yield
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Table 1 PNA sequences and DNA complements
Fig. 2 Temperature-dependent fluorescence spectra for PNA2 annealed
with DNA strands containing a single mismatch, either guanine (MMG,
), adenine (MMA, ), cytosine (MMC, ) or the matched complementary
᭹
strand containing thymine (T, ). Strand concentration is 2 mM in a buffer
containing NaCl (100 mM), EDTA (0.1 mM), and Na2PO4 (10 mM, pH
7). Fluorescence excitation at l = 312 nm and emission was measure at l =
349 nm.
(data not shown). The DAP monomer 9 was incorporated into
PNA1 and the 2AP monomer 13 into PNA2 (Table 1).
DNA binding studies
A PNA:DNA binding study was performed using PNA1 to
examine the effect of 2,6-diaminopurine on duplex stability. As
expected, the PNA1:DNA1T duplex exhibited the highest melting
temperature (62.5 ◦C), followed by the pyrimidine mismatch
PNA1:DNA1C melting at 56.5 ◦C. Due to the increased steric
bulk, the purine mismatches PNA1:DNA1G and PNA1:DNA1A
showedgreater decreases in helixstabilitywith Tm values of 49.5 ◦C
and 50 ◦C, respectively. Overall the DAP substitution showed a
mismatch selectivity of +6 ◦C, in this particular sequence context,
which is comparable with other DAP PNA:DNA studies.7
The binding study of PNA2 with DNA showed a surprising
selectivity for T of at least +12 ◦C. This is the first study of
the stabilization effect of 2AP in a PNA:DNA duplex, and
demonstrates its promise as a replacement for A.
Fig. 3 Fluorescence profile of PNA2 in single strand form and annealed
to DNA2B at 2 mM, in a buffer containing NaCl (100 mM), EDTA (0.1
mM), and Na2PO4 (10 mM) at pH 7. Excitation spectra (ex) are presented
in the range 200–330 nm and emission spectra (em) are presented in the
range 330–550 nm.
Fluorescence studies
Using 2AP, duplex denaturation was also examined by variable-
temperature fluorescence studies (Fig. 2). The results are com-
parable to those obtained by temperature-dependent UV-vis
spectroscopy and indicate that the changes in the environment
about the fluorescent 2AP are representative of global melting, for
this particular sequence.
The steady-state fluorescence excitation and emission spectra
were measured for single stranded PNA2 and PNA2:DNA2
duplexes (Fig. 3). G has been shown to stack with 2AP, creating a
charge-transfer complex which quenches fluorescence.16 However,
in the PNA2 sequence there is no adjacent intrastrand G. Rather,
when base paired to a G mismatch, the degree of fluorescence is
similar to the single strand which implies that 2AP is extrahelical,
a situation observed for this mismatch in DNA:DNA duplexes.17
The degree of fluorescence quenching parallels that of the
duplex stability. The most stable duplex is the perfectly matched
PNA2:DNA2T, which exhibits the greatest fluorescence quench-
ing, likely because it forms a stable stacked base pair within the
helix. The mismatched duplexes with 2AP across from C or A
likely possess bulged structures with correspondingly less stacking
and thus exhibited less fluorescence quenching.
Conclusion
A facile synthesis of Fmoc/Boc protected PNA monomers
has been established for thymine, 2,6-diaminopurine and
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2-aminopurine, which is compatible with standard Fmoc-based
oligomerization chemistry. In conjugation with our previous
report,5 there now exists a complete set of the natural nucleobases
and two useful analogues as the Fmoc/Boc monomers. The
per-Boc protected nucleobase monomers offers excellent organic
solvent solubility and orthogonality to hydrogenolysis that the
Bhoc- and Cbz-protected nucleobases do not.
The T PNA monomer was found to incorporate into PNAs
under standard coupling conditions as well as the commercially
available monomer with no evidence of truncation for a T5 tract.
The DAP monomer was incorporated into a PNA, and in a binding
study with DNA, it was found to selectively bind to T (+6 ◦C). The
2AP monomer showed excellent discrimination of T (+12 ◦C) over
the other nucleobases and also acted as a fluorescent probe of the
PNA:DNA duplexes. A fully matched duplex was characterized by
the greatest degree of fluorescence quenching whereas mismatches
displayed less quenching. The severity of the mismatch, as judged
by the degree of helix destabilization determined by the UV-
vis measured Tm values (G > A > C), is qualitatively inversely
correlated with the degree of quenching (C > A > G). These results
strongly suggest that 2AP is a base discriminating fluorophore in
PNA:DNA duplexes, much in the same way as it is in dsDNA,
and may find similar uses in the study of PNA:DNA structures
and dynamics.
128.2, 110.5, 86.6, 67.5, 48.6, 27.2, 12.1; HRMS (EI) calculated
for [C19H22N2O6]+: 374.1478, found 374.1484.
N3-Boc-thyminyl-1-acetic acid (2). 1 (0.75 g, 2.0 mmol) was
dissolved in minimal acetone : MeOH solution (1 : 1, v/v). Pd/C
(80 mg) was added and the reaction flask was placed under an
atmosphere of H2. The suspension was stirred at room temperature
for 2 h. The solvent was removed and the residue suspended in
MeOH (100 mL). The suspension was filtered through Celite, and
the solvent removed to yield 2 (0.57 g, quant. yield) as a glassy
white solid: 1H NMR (400 MHz, DMSO-d6) = 7.66 (s, 1 H), 4.43
(s, 2 H), 1.81 (s, 3 H), 1.51 (s, 9 H); 13C NMR (101 MHz, DMSO-
d6) = 169.8, 164.5, 151.1, 142.0, 141.8, 108.4, 49.1, 48.7, 27.7,
12.0; HRMS (EI) calculated for [C11H16N2O6]+: 284.1008, found
284.1021.
N3-Boc-thyminyl-1-acetate Fmoc/Bn PNA backbone (3).
Fmoc/Bn PNA backbone (0.927 g, 2.13 mmol) and 2 (0.759 g, 2.67
mmol) were dissolved in DCM (10 mL) and cooled to 0 ◦C. EDC
(0.497 g, 3.20 mmol) and HOBt hydrate (0.081 g, 0.53 mmol) were
added. The solution was stirred for 0.5 h then allowed to warm
to room temperature. After 18 h a saturated NaHCO3 solution
(100 mL) was added and extracted with DCM (2 ¥ 100 mL). The
organic layers were dried over Na2SO4 and the solvent removed.
The residue was purified by FCC (EtOAc : hexane = 8 : 2 to 9 : 1)
to yield 3 (1.14 g, 77% yield) as a white foam: 1H NMR (400 MHz,
DMSO-d6) = 7.88 (d, J = 7.4 Hz, 2 H), 7.67 (d, J = 7.8 Hz, 2 H),
7.26–7.48 (m, 10 H), 5.21 (s, min.), 5.13 (s, maj.), 4.76 (s, 2 H),
4.58 (s, min.), 4.10–4.40 (m, 5 H), 2.99–3.61 (m, 5 H), 1.79 (s, 3
H), 1.48 (s, 9 H); 13C NMR (101 MHz, CDCl3) = 170.7, 169.0,
168.8, 167.2, 166.9, 161.4, 161.3, 156.4, 156.3, 148.7, 148.6, 147.7,
143.5, 143.4, 140.8, 140.5, 140.3, 134.7, 134.4, 128.2, 128.1, 128.1,
127.9, 127.8, 127.4, 127.3, 126.7, 124.7, 119.6, 109.7, 109.5, 86.3,
86.2, 67.4, 66.8, 66.4, 66.2, 60.0, 49.8, 48.5, 48.2, 47.9, 47.6, 47.5,
46.7, 38.8, 38.4, 27.0, 20.6, 13.8, 11.9; HRMS (EI) calculated for
[C38H40N4O9Na]+: 719.2693, found 719.2693.
Experimental
General
All chemicals were obtained from commercial sources and were
of ACS reagent grade or higher and were used without further
purification. Solvents for solution-phase chemistry were dried by
passing through columns of activated alumina. Flash column
chromatography (FCC) was performed on Merck Kieselgel 60,
230–400 mesh. Thin layer chromatography (TLC) was performed
on Merck Kieselgel 60 TLC plates. Chemical shifts (d) are reported
in parts per million (ppm), were measured from tetramethylsilane
(0 ppm) and are referenced to the solvent CDCl3 (7.26 ppm),
DMSO-d6 (2.49 ppm), D2O (4.79 ppm) for 1H NMR and CDCl3
(77.0 ppm), DMSO-d6 (39.5 ppm) for 13C NMR. Multiplicities
are described as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet) and br s (broad singlet). Coupling constants (J)
are reported in Hertz (Hz). Resonances due to restricted rotation
around the amide bond (rotamers) are reported as major (ma.) and
minor (mi.). High resolution mass spectra (HRMS) were obtained
using electron impact (EI) or electrospray ionization (ESI).
N3-Boc-thymin-1-yl acetate Fmoc PNA monomer (4). 3 (0.251
g, 0.36 mmol) was dissolved in the minimal amount of an
acetone : MeOH solution (1 : 1, v/v). Pd/C (80 mg) was added and
the reaction flask was placed under an atmosphere of H2 at 0 ◦C.
After 0.5 h the suspension was stirred at room temperature for a
further 2 h. The solvent was removed and the residue suspended in
MeOH (100 mL). The suspension was filtered through Celite, and
the solvent removed to yield 4 (0.212 g, 95% yield) as an off-white
1
powder: H NMR (400 MHz, DMSO-d6) = 12.79 (s, 1 H), 7.89
(d, J = 7.4 Hz, 2 H), 7.68 (d, J = 7.4 Hz, 2 H), 7.10–7.52 (m, 5
H), 4.74 (s, 1 H), 4.55 (s, 1 H), 4.14–4.39 (m, 3 H), 4.00 (s, 1 H),
3.19–3.47 (m, 5 H), 3.03–3.17 (m, 1 H), 1.79 (s, 3 H), 1.49 (s, 9
H);13C NMR (151 MHz, DMSO-d6) = 171.0, 170.6, 167.8, 167.4,
166.8, 164.5, 161.4, 156.5, 156.3, 151.1, 148.6, 148.1, 144.0, 142.1,
140.8, 127.7, 127.2, 125.2, 120.2, 108.3, 86.2, 65.7, 47.9, 47.1, 46.9,
31.4, 27.1, 12.0; HRMS (ESI) calculated for [C31H34N4O9Na]+:
629.2223, found 629.2252.
Benzyl N3-Boc-thymin-1-yl acetate (1). Benzyl thyminyl-1-
acetate (3.0 g, 11 mmol), Boc2O (4.8 g, 22 mmol), and DMAP (2.7
g, 22 mmol) were added to THF (30 mL) at 0 ◦C. After 0.5 h the
solution was warmed to room temperature. After 18 h, the reaction
mixture was concentrated in vacuo. The residue was dissolved in
DCM (100 mL) and extracted with H2O (3 ¥ 100 mL). The organic
layer was dried over anhydrous Na2SO4 and the solvent removed.
The residue was purified by FCC (EtOAc : hexane = 2 : 3, v/v)
to give 1 (3.9 g, 95% yield) as a light-yellow foam: 1H NMR (400
MHz, CDCl3) = 7.30–7.41 (m, 5 H), 6.90 (s, 1 H), 5.20 (s, 2 H), 4.47
(s, 2 H), 1.92 (s, 3 H), 1.56–1.62 (m, 9 H);13C NMR (101 MHz,
CDCl3) = 167.1, 161.4, 148.8, 147.6, 139.7, 134.6, 128.5, 128.4,
Penta(Boc)-2,6-diaminopurine (5). 2,6-Diaminopurine (0.500
g, 3.33 mmol), Boc2O (6.12 g, 26.6 mmol), and DMAP (0.061 g,
0.50 mmol) were added to THF (25 mL) at 0 ◦C. The solution
was stirred for 5 min then warmed to room temperature. After 18
h the solvent was removed. The residue was dissolved in DCM
(150 mL) and washed with H2O (100 mL). The organic layer was
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dried over Na2SO4 and the solvent was removed. The residue was
purified by FCC (EtOAc : hexane = 1 : 2) to yield 5 (2.08 g, 96%
yield) as a white foam: 1H NMR (400 MHz, CDCl3) = 8.52 (s, 1 H),
1.68 (s, 9 H), 1.43 (s, 18 H), 1.41 (s, 18 H); 13C NMR (101 MHz,
CDCl3) = 153.7, 152.9, 151.6, 150.6, 149.7, 145.5, 143.8, 127.8,
87.3, 83.9, 83.2, 41.9, 27.8, 27.7, 27.6; HRMS (EI) calculated for
[C30H46N6O10]+: 650.3275, found 650.3262.
7.49 (m, 9 H), 5.41 (br s, 1 H), 5.23 (br s, 2 H), 5.10 (s, 1 H), 4.50
(s, 1 H), 4.36 (d, J = 7.0 Hz, 1 H), 4.13–4.32 (m, 3 H), 3.50–3.60
(m, 1 H), 3.06–3.16 (m, 1 H), 1.28–1.37 (m, 36 H);13C NMR (101
MHz, CDCl3) = 154.6, 152.2, 151.1, 151.1, 150.2, 147.1, 144.0,
143.8, 141.5, 129.2, 129.1, 128.9, 128.8, 128.5, 128.0, 128.0, 127.3,
125.3, 125.1, 120.3, 84.0, 83.6, 67.6, 66.7, 49.4, 49.2, 47.4, 31.8,
28.0, 27.9, 22.9, 14.4; HRMS (ESI) calculated for [C53H65N8O13]+:
1021.4671, found 1021.4711.
Tetra(Boc)-9H-2,6-diaminopurine (6). 5 (1.93 g, 2.97 mmol)
was suspended in mixture of MeOH (50 mL) and saturated
NaHCO3 solution (25 mL). The turbid solution was heated to
Tetra(Boc)-2,6-diaminopurin-9-yl acetate Fmoc PNA monomer
(10). 9 (0.796 g, 0.78 mmol) was dissolved in MeOH (20 mL) and
Pd/C (50 mg) was added. The reaction vessel was placed under
an atmosphere of H2. The suspension was stirred for 3 h at room
temperature. The reaction was filtered through Celite with MeOH
(100 mL) and the solvent removed to yield 10 (0.726 g, 96% yield)
as a flaky white solid: 1H NMR (400 MHz, acetone-d6) = 8.31 (s,
1 H), 7.75–7.83 (m, 2 H), 7.63 (d, J = 7.4 Hz, 2 H), 7.22–7.39 (m,
4 H), 5.38 (s, 1 H), 5.26 (s, 1 H), 4.34–4.44 (m, 2 H), 4.09–4.29 (m,
3 H), 3.69 (t, J = 6.3 Hz, 1 H), 3.43–3.55 (m, 2 H), 3.25–3.33 (m,
1 H), 1.30–1.42 (m, 36 H); 13C NMR (101 MHz, acetone-d6) =
171.0, 167.9, 167.3, 157.5, 155.8, 152.8, 151.7, 151.2, 151.0, 148.9,
145.1, 142.1, 128.6, 128.0, 128.0, 127.3, 126.2, 126.1, 120.9, 84.0,
83.5, 67.0, 48.1, 45.3, 40.0, 28.0, 27.9; HRMS (ESI) calculated for
[C46H59N5O13]+: 931.4196, found 931.3406.
◦
50 C for 1 h and then cooled to room temperature. After 18 h
the MeOH was removed by rotary evaporation and the solution
was diluted with H2O (100 mL) and extracted with DCM (2 ¥ 100
mL). The combined organic layers were dried over Na2SO4 and
the solvent was removed to yield 6 (1.12 g, 68% yield) as a white
1
foam: H NMR (400 MHz, CDCl3) = 8.63 (s, 1 H), 5.05 (br s, 2
H), 1.36 (s, 18 H), 1.33 (s, 18 H);13C NMR (101 MHz, CDCl3) =
151.4, 150.8, 149.9, 145.3, 109.9, 84.3, 83.4, 27.7, 27.6; HRMS
(EI) calculated for [C25H38N6O8]+: 550.2751, found 550.2757.
Benzyl tetra(Boc)-2,6-diaminopurin-9-yl acetate (7). 6 (0.500
g, 0.908 mmol) and K2CO3 (0.138 g, 0.999 mmol) were added to
DMF (3 mL) at 0 ◦C. Benzyl bromoacetate (0.16 mL, 1.0 mmol)
was added dropwise over 5 min. The solution was stirred for 10
min then warmed to room temperature. After 18 h the reaction
mixture was diluted with H2O (100 mL) and extracted with ether
(2 ¥ 100 mL). The organic layers were combined, washed with
water (50 mL), dried over Na2SO4, and the solvent removed to
yield 6 (0.616 g, 97% yield) as a white foam: 1H NMR (400 MHz,
CDCl3) = 8.14 (s, 1 H), 7.31–7.40 (m, 5 H), 5.21 (s, 2 H), 5.05 (s, 2
H), 1.40 (s, 18 H), 1.39 (s, 18 H); 13C NMR (101 MHz, CDCl3) =
176.0, 167.0, 154.2, 152.2, 151.0, 150.6, 149.8, 145.8, 128.7, 128.4,
126.7, 83.7, 83.2, 67.9, 52.9, 44.3, 44.1, 27.7, 27.6; HRMS (ESI)
calculated for [C34H47N6O10]+: 699.3348, found 699.2743.
Benzyl 6-chloro-2-diBoc-aminopurin-9-yl acetate (11a). 6-
Chloro-2-diBoc-aminopurine (1.28 g, 3.45 mmol) and K2CO3
(0.524 g, 3.79 mmol) were suspended in DMF (15 mL) and cooled
to 0 ◦C. Benzyl bromoacetate (0.60 mL, 3.8 mmol) was added
dropwise over 10 min. The solution was stirred for 5 min and
then warmed to room temperature. After 18 h the suspension was
filtered through Celite with EtOAc (100 mL) then concentrated
in vacuo. The oil obtained was suspended in H2O (100 mL)
and extracted with Et2O (2 ¥ 200 mL). The organic layers were
combined, dried over Na2SO4 and the solvent removed. The
residue was purified by FCC (EtOAc : Hex = 1 : 1) to yield 11 (1.32
Tetra(Boc)-2,6-diaminopurin-9-yl acetic acid (8). 7 (0.509 g,
0.728 mmol) was dissolved in MeOH (5 mL). Pd/C (60 mg) was
added and the reaction vessel was placed under an atmosphere
of H2. The suspension was stirred for 2 h at room temperature.
The suspension was filtered through Celite with MeOH (100 mL)
and the solvent removed to yield 8 (0.439 g, 99% yield) as a glassy
1
g, 74% yield) as a white powder: H NMR (400 MHz, CDCl3) =
8.19 (s, 1 H), 7.31–7.41 (m, 5 H), 5.22 (s, 2 H), 5.05 (s, 2 H), 1.42
(s, 18 H); 13C NMR (101 MHz, CDCl3) = 166.2, 152.7, 152.0,
151.1, 150.3, 146.4, 134.2, 129.4, 128.8, 128.6, 128.5, 83.6, 68.1,
44.4, 27.7; HRMS (ESI) calculated for [C24H29ClN5O6]+: 518.1801,
found 518.1806.
1
grey solid: H NMR (400 MHz, DMSO-d6) = 8.63 (s, 1 H), 5.05
(br s, 2 H), 1.35 (s, 18 H), 1.33 (s, 18 H); 13C NMR (101 MHz,
MeOH-d4) = 200.7, 156.1, 153.2, 152.4, 151.5, 150.0, 85.6, 85.1,
28.2, 28.1; HRMS (ESI) calculated for [C27H41N6O10]+: 609.2879,
found 609.2688.
2-Di(Boc)-aminopurin-9-yl acetic acid (12). 11a (0.304 g, 0.587
mmol) was dissolved in a mixture of acetone (4 mL), Et3N (1.4
mL), and formic acid (0.2 mL). Pd/C (40 mg) was added and
the reaction mixture was brought to reflux. The two products
formed were tracked by HPLC. Two further equivalents of Pd/C
(40 mg), Et3N (1.4 mL), and formic acid (0.2 mL) were added
after 2 h and 4 h. When only 1 product remained by HPLC, the
mixture was filtered through Celite with acetone (100 mL) and
the solvent removed. The residue was suspended in H2O (25 mL)
and extracted with DCM (3 ¥ 50 mL). The organic fractions were
combined and washed with a 1 M KHSO4 solution (50 mL). The
organic layer was dried over Na2SO4 and the solvent removed to
Tetra(Boc)-2,6-diaminopurin-9-yl acetate Fmoc/Bn PNA back-
bone (9). Fmoc/Bn PNA backbone (0.061 g, 0.180 mmol), 8
(0.164 g, 0.270 mmol), EDC (0.056 g, 0.36 mmol) and HOBt
hydrate (0.006 g, 0.04 mmol) were added to DCM (5 mL) at 0 ◦C.
The solution stirred for 20 min then warmed to room temperature.
The reaction was monitored by TLC. Upon completion, the
reaction mixture was diluted with DCM (100 mL) and washed
against a mixture of a saturated NaHCO3 solution (25 mL)
and brine (50 mL). The organic layer was dried over Na2SO4,
and the solvent removed. The residue was subjected to FCC
(EtOAc : hexane = 2 : 1 to 1 : 0) to yield 9 (0.131 g, 71% yield) as
a white powder: 1H NMR (400 MHz, DMSO-d6) = 8.49 (s, min.),
8.47 (s, maj.), 7.83–7.89 (m, 2 H), 7.66 (t, J = 6.3 Hz, 2 H), 7.25–
1
yield 12 (0.186 g, 81% yield) as a glassy white solid: H NMR
(400 MHz, MeOH-d4) = 9.10 (s, 1 H), 8.57 (s, 1 H), 5.17 (s, 2 H),
1.39 (s, 18 H); 13C NMR (101 MHz, MeOH-d4) = 170.0, 154.4,
153.9, 152.2, 150.3, 149.8, 133.3, 85.0, 45.3, 28.2; HRMS (ESI)
calculated for [C17H24N5O6]+: 394.1721, found 394.1727.
880 | Org. Biomol. Chem., 2012, 10, 876–881
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2-Di(Boc)-aminopurin-9-yl acetate Fmoc/Bn PNA (13).
Fmoc/Bn PNA backbone (0.276 g, 0.81 mmol), 12 (0.264 g, 0.67
mmol), EDC (0.155 g, 1.00 mmol) and HOBt hydrate (0.026 g,
0.17 mmol) were added to DCM (5 mL) at 0 ◦C. The solution
stirred for 40 min and then warmed to room temperature. After 2
days the reaction was diluted with DCM (150 mL), washed with
a saturated NaHCO3 solution (100 mL), H2O (100 mL) and brine
(100 mL). The organic layer was dried over Na2SO4 and the solvent
removed. The residue was subjected to FCC (EtOAc : Hex = 4 : 1
to 1 : 0) to yield 13 (0.313 g, 58% yield) as a white foam: 1H NMR
(400 MHz, DMSO-d6) = 9.17 (s, 1 H), 8.47 (d, 1 H), 7.87 (d, J =
7.2 Hz, 2 H), 7.66 (t, J = 7.5 Hz, 2 H), 7.22–7.52 (m, 9 H), 5.40 (br
s, 1 H), 5.23 (br s, 1 H), 5.09 (s, 1 H), 4.50 (br s, 1 H), 4.10–4.40
(m, 4 H), 3.51–3.60 (m, 1 H), 3.07–3.15 (m, 1 H), 1.31 (s, 18 H);
13C NMR (101 MHz, CDCl3) = 169.3, 156.6, 152.7, 151.0, 149.3,
147.4, 146.4, 143.7, 143.5, 141.2, 128.8, 128.6, 128.6, 128.2, 127.7,
127.7, 127.0, 124.8, 120.0, 109.9, 83.3, 67.4, 49.2, 47.1, 29.6, 27.8,
27.3; HRMS (ESI) calculated for [C43H48N7O9]+: 806.3514, found
806.3529.
Melting temperature (Tm) measurements
All Tm measurements of the PNA:DNA duplexes (2 mM concen-
tration) were made in a 10 mM sodium phosphate buffer (pH
7.0) containing 100 mM NaCl and 0.1 mM EDTA. Absorbance
vs. temperature profiles were measured using a Cary 300-Bio
spectrophotometer using a cell with a 1 cm path length. The
absorbance of the sampl◦es was monitored at 260 nm from 15 ◦C
to 85 ◦C, at a rate of 0.5 C min-1.
Notes and References
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2-Di(Boc)-aminopurin-9-yl acetate Fmoc PNA monomer (14).
13 (0.313 g, 0.389 mmol) was dissolved in MeOH (10 mL) and
Pd/C (100 mg) was added. The reaction mixture was placed
under an atmosphere of H2. The suspension was stirred for 12 h
at room temperature. The suspension was filtered through Celite
with MeOH (100 mL) and the solvent removed to yield 14 (0.266
g, 95% yield) as an off-white solid: 1H NMR (400 MHz, CDCl3) =
8.99–9.21 (m, 2 H), 8.23–8.39 (m, maj.), 8.10 (br s, min.), 7.62–
7.81 (m, 2 H), 7.44–7.62 (m, 2 H), 7.11–7.41 (m, 4 H), 6.83 (br
s, maj.), 6.05 (br s, min.), 4.96–5.16 (m, 1 H), 4.85 (br s, 1 H),
4.29–4.42 (m, 1 H), 4.11–4.27 (m, 2 H), 4.01 (br s, 1 H), 3.77
(br s, 1 H), 3.51 (br s, 2 H), 3.26–3.42 (m, 1 H), 2.74 (br s, 2
H), 1.44 (s, 18 H); 13C NMR (400 MHz, CDCl3) = 171.6, 166.9,
166.3, 156.9, 152.7, 151.0, 148.9, 148.3, 143.6, 141.2, 131.4, 127.7,
127.1, 125.0, 124.3, 120.0, 83.6, 66.7, 50.6, 48.9, 47.1, 39.3, 27.8;
HRMS (ESI) calculated for [C36H41N7O9Na]+: 738.2863, found
738.2877.
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