A Fmoc/Boc pseudoisocytosine monomer for peptide nucleic acid synthesis

Article abstract of DOI:10.1139/V08-144

The synthesis of N-[2-(fluorenylmethoxycarbonyl)aminoethyl]-N-[(2-N-(tert- butoxycarbonyl)isocytosin-5-yl)acetyl]glycine peptide nucleic acid monomer is described. The N-tert-butoxycarbonyl protected monomer is compatible with commercially available monomers designed for the Fmoc-oligomerization strategy and is deprotected under mild acidolysis conditions.

Full text of DOI:10.1139/V08-144

1026
A Fmoc/Boc pseudoisocytosine monomer for
peptide nucleic acid synthesis
Filip Wojciechowski and Robert H.E. Hudson
Abstract: The synthesis of N-[2-(fluorenylmethoxycarbonyl)aminoethyl]-N-[(2-N-(tert-butoxycarbonyl)isocytosin-5-
yl)acetyl]glycine peptide nucleic acid monomer is described. The N-tert-butoxycarbonyl protected monomer is compati-
ble with commercially available monomers designed for the Fmoc-oligomerization strategy and is deprotected under
mild acidolysis conditions.
Key words: PNA, polyamide, pseudoisocytosine, J-base, triplex.
Résumé : On décrit la synthèse du peptide monomère de l’acide nucléique N-[2-(fluorénylméthoxycarbonyl)amino-
éthyl]-N-[2-N-(tert-butoxycarbonyl)isocytosin-5-yl)acétyl]glycine. Le monomère protégé par le groupe N-tert-
butoxycarbononyle est compatible avec les monomères disponibles commercialement, développés pour la stratégie
d’oligomérisation Fmoc, et il peut être déprotégé dans des conditions d’acidolyse douce.
Mots-clés : PNA, polyamide, pseudoisocytosine, base J, triplex.
[Traduit par la Rédaction] Wojciechowski and Hudson 1029
Introduction
other nucleobases that have shown beneficial properties are
not, and synthetic procedures for N-protection with mild
acid labile protecting groups have not been thoroughly in-
vestigated.
Peptide nucleic acid (PNA) is a nucleic acid analogue
composed of a polyamide backbone to which the nucleo-
bases are attached by a methylene carbonyl group (1). PNA
oligomers hybridize to complementary DNA or RNA obey-
ing Watson–Crick base-pairing rules and do so with remark-
able sequence specificity and high affinity. Interest in PNAs
as therapeutic agents (2) or as probes in clinical diagnostics
(3) continues to motivate scientists to expand the frontiers of
PNA chemistry and its applications. It is evident that such
uses will only flourish if the building blocks for the synthe-
sis of modified PNA are commercially available or readily
prepared by straightforward synthetic procedures. Thus it
becomes important to develop and detail the route to such
reagents to benefit the community interested in PNA.
Although there have been a number of nucleobase modifi-
cations incorporated into PNAs (4), the majority of these
nucleobases utilize Boc/Cbz-protected monomers. Removal
of the Cbz exocyclic amino protecting group from the
nucleobases requires harsh reagents, usually neat HF or
TFMSA, and is generally disfavoured by the nonspecialist.
Therefore, other protecting group strategies for mild
deprotection have been investigated.
Nucleic acids (NAs) with purine-consecutive sequences
may be targeted by homopyrimidine PNA forming a
(PNA)2:NA triplex that has a very high thermal stability (5).
One PNA strand engages in Watson–Crick base-pairing,
while the other PNA strand is involved in Hoogsteen bind-
ing. While thymine forms a stable Hoogsteen pair with ade-
nine, cytosine requires protonation below physiological
conditions to form a stable Hoogsteen pair with guanine.
Such pH dependence may be alleviated by using nucleobase
analogues of protonated cytosine such as N7-guanine (6) or
pseudoisocytosine (J) (7) (Fig. 1). To date, neither a syn-
thetic procedure nor a commercial source for pseudoiso-
cytosine protected by a mild acid labile protecting group has
been reported.
We have recently developed a convenient route to benzyl,
allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)amino-
ethyl]glycine to facilitate the syntheses of PNA monomers in
which the nucleobases are protected with groups that could
be liberated under mild acidic conditions. We have also suc-
cessfully synthesized PNA oligomers using a bis-N-Boc
nucleobase protecting group strategy to reduce aggregation
during oligomer synthesis (8). Herein we report the first syn-
thesis and oligomerization of a pseudoisocytosine monomer
protected at the exocyclic amino group with a mild acid la-
bile protecting group (Boc), which is fully compatible with
standard Fmoc/Bhoc oligomerization–deprotection peptide
synthesis.
Currently, the most commonly used PNA building blocks
for mild deprotection are the commercially available
Fmoc/Bhoc monomers. While Bhoc-protected adenine, thy-
mine, guanine, and cytosine are commercially available,
Received 21 June 2008. Accepted 2 August 2008. Published
on the NRC Research Press Web site at canjchem.nrc.ca on
8 October 2008.
F. Wojciechowski and R.H.E. Hudson.1 Department of
Chemistry, The University of Western Ontario, London, ON
N6A 5B7, Canada.
Results and discussion
The synthesis of isocytosin-5-ylacetic acid (9) (1) was
carried out as described by the modified procedure of Niel-
1Corresponding author: (e-mail: rhhudson@uwo.ca).
Can. J. Chem. 86: 1026–1029 (2008)
doi:10.1139/V08-144
© 2008 NRC Canada

Wojciechowski and Hudson
1027
Scheme 1. Synthesis of N2-(tert-butoxycarbonyl)isocytosin-5-
ylacetic acid 4. Reagents and conditions: (a) HCl(g), EtOH,
reflux 2 h; (b) (Boc)2O, DMAP, DMF, 36 h; (c) NaOH,
THF/H2O, 15 min.
Fig. 1. Nucleobase interactions for a (PNA)2:NA triple helix.
(a) Protonated cytosine (C+) and the G:C base pair (C+:G:C) and
(b) pseudoisocytosine (J) and the G:C base pair (J:G:C).
(Scheme 1). Alternatively, acidification of the carboxylate of
4 with 4 mol/L of HCl followed by extraction into ethyl ace-
tate resulted in a lower yield (73%) and required the use of
copious amounts of ethyl acetate (400 mL) because of the
poor solubility of 4 in ethyl acetate.
sen and co-workers (10). Condensation of dimethyl suc-
cinate with methyl formate gave methyl α-formylsuccinate
as a colourless oil that was partially contaminated with
dimethyl succinate. The product may be isolated free of
dimethyl succinate by continuous extraction with diethyl
ether but this greatly reduces the yield. Nielsen and co-
workers proposed that this contaminated oil may be directly
used in the next step. Therefore, the crude mixture was re-
acted with guanidine hydrochloride to give isocytosin-5-
ylacetic acid 1 in good yield, confirming that it is suitable to
use the crude mixture. Subsequent esterification with
EtOH/HCl(g) gave ethyl isocytosin-5-ylacetate hydrochlo-
ride, which was dissolved in aqueous sodium bicarbonate
and acidified to pH 6.0 to give ethyl isocytosin-5-ylacetate
(2) in 81% yield.
Carbodiimide-mediated coupling of
4
with N-[2-
(Fmoc)aminoethyl]glycine benzyl ester (8) was accom-
plished using EDC/HOBt, to give the orthogonally protected
monomer ester 5 in 74% yield. Removal of the benzyl ester
using Pd/C and H2 gave the desired monomer 6 in 86% yield
(Scheme 2).
The performance of the Boc-protected pseudoisocytosine
monomer (6, J) was judged by incorporation into a 10-mer
PNA sequence (N-term: GTA GAT CJC T-Lys C-term, 7) by
standard Fmoc-mediated peptide synthesis along with com-
mercially available Bhoc-protected monomers. The coupling
of monomer 6 was qualitatively judged to be trouble-free by
conductivity monitoring during the automated synthesis of
the oligomer. Furthermore, RP-HPLC analysis of the crude
oligomer (Fig. 2) indicated the coupling to be equivalent to
the standard G, T, C, and A monomers with an average step-
wise coupling yield of >98.5%.
Post oligomerization, removal of the Boc protecting group
along with the Bhoc protecting groups was achieved using
TFA/Et3SiH (975:25 µL). The crude oligomer was purified
by RP-HPLC; the major peak in the chromatogram corre-
sponded to the full length oligomer (retention time:
28.375 min.). The identity of 7 was confirmed by mass spec-
trometry (Fig. 3). There was excellent agreement between
the expected and found masses: TOF-MS (ESI) m/z calcd.
for C113H148N58O32: 2830.75; found: 2830.80.
In summary, we report the synthesis of a pseudo-
isocytosine PNA monomer that possesses a Boc protecting
group on the nucleobase and its subsequent incorporation
into a PNA oligomer by Fmoc-based chemistry. Previous at-
tempts at protection of pseudoisocytosine with acid labile
protecting groups have resulted in poor yields (11) without
reporting the experimental procedures. Thus, this is the first
We have previously reported the bis-N-Boc protection of
ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate, and ethyl
(O6-benzylguanin-9-yl)acetate. The exocyclic amino group
of cytosine was most reactive towards (Boc)2O and gave the
bis-N-Boc protected nucleobase in excellent yield. Likewise
we reasoned that pseudoisocytosine, which is a cytosine ana-
logue, should be prone to bis-N-Boc protection. Thus, ethyl
isocytosin-5-ylacetate 2 was reacted with a catalytic amount
of DMAP, 4.0 equiv. of (Boc)2O, triethylamine in CH3CN
under reflux, but the major product was the mono-Boc pro-
tected
derivative
N2-(tert-butoxycarbonyl)isocytosin-5-
ylacetate (3) in 51% yield. Changing to N-methylimidazole
as the catalyst, reducing the amount of (Boc)2O to 1.3 equiv.,
and changing to DMF as the solvent gave 3 in slightly better
yield (57%). Final optimization gave 3 in 70% yield free of
column chromatography, by simply using a catalytic amount
of DMAP and 1.3 equiv. of (Boc)2O in DMF. Subsequent
hydrolysis of 3 gave the carboxylate, which was acidified
with citric acid to give a precipitate of N2-(tert-butoxy-
carbonyl)isocytosin-5-ylacetic acid (4) in 91% yield
© 2008 NRC Canada

1028
Can. J. Chem. Vol. 86, 2008
Scheme 2. Synthesis of Boc protected pseudoisocytosine mono-
mer 6. Reagents and conditions: (a) EDC/HOBt, DMF, 36 h;
(b) Pd/C, H2, MeOH, 3 h.
Fig. 3. Transformed data for the ESI-TOF mass spectral analysis
of the 10-mer PNA.
Fig. 2. Reversed-phase HPLC analysis of crude oligomer 7: GTA
GAT CJC T-Lys.
(ma.) and minor (mi.). Coupling constants (J) are reported in
Hertz (Hz). High-resolution mass spectra (HRMS) were ob-
tained using electron impact (EI) or electrospray (ESI).
Ethyl isocytosin-5-ylacetate (2)
To ethanol (150 mL) was added isocytosin-5-ylacetic acid
1 (4.5 g, 26.6 mmol) and HCl(g) in ethanol (50 mL,
1.5 mol/L). The mixture was heated to reflux, and within
15 min the mixture became homogeneous and the solution
was refluxed for a further 2 h. The solution was cooled to
room temperature, concentrated in vacuo to approximately
100 mL, and placed at –20 °C for 2 h. The precipitate was
collected by filtration and washed with diethyl ether to give
a white solid. The hydrochloride salt of ethyl isocytosin-5-
ylacetate was dissolved in saturated aqueous sodium bicar-
bonate (50 mL), and then acidified to pH 6.0 by dropwise
addition of 4 mol/L of HCl while being stirred. The precipi-
tate was cooled at 4 °C for 1 h, collected by filtration, and
washed with diethyl ether to give a white solid (4.2 g, 81%).
1H NMR (400 MHz, DMSO-d6) δ: 10.98 (br s, 1H), 7.46 (s,
1H), 6.47 (br s, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.19 (s, 2H),
1.16 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ:
171.2, 164.7, 156.0, 151.5, 108.7, 60.0, 32.1, 14.1. HRMS
(EI) calcd. for C8H11N3O3: 197.0800 [M+]; found: 197.0809.
report for such a protection and will facilitate its future use
in triplex forming PNA, assembled by Fmoc-based peptide
synthesis.
Experimental
General
All reagents and solvents were obtained from commercial
sources, were of ACS reagent grade or higher, and were
used without further purification. Anhydrous solvents were
prepared by passing the reagent-grade solvent through col-
umns of activated alumina (Innovative Technologies). 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 are reported in parts per million (δ), were
measured from tetramethylsilane (0 ppm), and are refer-
enced to the residual proton in the otherwise perdeuterated
N2-(tert-butoxycarbonyl)isocytosin-5-ylacetate (3)
A suspension of 2 (1.0 g, 5.0 mmol), DMAP (61 mg,
0.5 mmol), and Boc2O (1.4 g, 6.4 mmol) in DMF (5 mL)
was stirred for 48 h under N2. To the solution was added eth-
anol (5 mL), followed by saturated aqueous sodium bicar-
bonate (25 mL), and the solution was stirred for 10 min. The
mixture was extracted with EtOAc (4 × 50 mL), and the
combined organic extracts were subsequently washed with
brine (50 mL) and dried over Na2SO4. After removal of the
solvent, the residue was triturated with equal volumes of di-
ethyl ether/hexanes (50 mL) and collected by filtration to
1
solvents: CDCl3 (7.26 ppm), DMSO-d6 (2.49 ppm) for H
NMR and CDCl3; (77.0 ppm), DMSO-d6 (39.5 ppm) for 13C
NMR. Multiplicities are described as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad singlet (br s).
Resonances attributed to conformers due to restricted rota-
tion around amide bonds (rotamers) are reported as major
1
give 3 as a white solid (1.05 g, 70%). H NMR (400 MHz,
DMSO-d6) δ: 11.28 (br s, 2H), 7.67 (s, 1H), 4.03 (q, J = 7.0
© 2008 NRC Canada

Wojciechowski and Hudson
1029
Hz, 2H), 3.31 (s, 2H), 1.48 (s, 9H), 1.17 (t, J = 7.0 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ: 170.1, 160.2, 153.1, 151.4,
151.1, 115.1, 84.0, 60.6, 32.0, 27.7, 13.8. HRMS (EI) calcd.
for C13H19N3O5: 297.1325 [M+]; found: 297.1330.
7H), 1.41 (s,
9
H). HRMS (ESI-TOF) calcd. for
C30H33N5O8: 614.2227 [MNa+]; found: 614.2236.
Synthesis of PNA
The 10-mer PNA oligomer was synthesized on a 5 umol
scale using the standard FastMoc module on an Applied
Biosystems 433A synthesizer. A NovaSyn TGR (Rink am-
ide) resin preloaded with lysine (approximate loading of
0.065 mmol/g) was used. The resin bound oligomer (approx.
20 mg) was placed in a 2 mL screw top vial to which was
added an ice-cold solution of the deprotection “cocktail”
(TFA/Et3SiH 975 uL:25 uL). The resin was agitated for
1.5 h. Filtration through a Pasteur pipette packed with glass
wool (0.5 cm) into a clean 2 mL vial followed by removal of
the TFA with a gentle stream of nitrogen and precipitation
by the addition of cold diethyl ether (1 mL) gave an off-
white solid. The crude oligomer was dissolved in aq. TFA
(0.05%), filtered (0.45 µm), and purified by RP-HPLC using
a 250 mm × 4.6 mm, C18-bonded phase, 300 Å pore, and
5 µm particle size column. Mass spectral analysis of the
oligomer was performed at the UWO Biological Mass Spec-
trometry Laboratory using ESI-TOF MS.
N2-(tert-butoxycarbonyl)isocytosin-5-ylacetic acid (4)
A suspension of compound 3 (1.0 g, 3.4 mmol) in water/
THF (15 mL:5 mL) was placed in an ice bath, followed by
the dropwise addition of NaOH (2.5 mol/L, 10 mL) over
1 min. The suspension was removed from the ice bath and
stirred until TLC analysis indicated complete consumption
of the starting material (15 min). The precipitate was cooled
with the aid of an ice bath, and citric acid monohydrate
(5.3 g, 25.0 mmol) dissolved in water (4 mL) was added in
one portion. The mixture was stirred on ice for 1 h, and the
solid precipitate was collected by filtration and washed with
1
diethyl ether to give a white solid (830 mg, 91%). H NMR
(400 MHz, DMSO-d6) δ: 11.60 (br s, 3H), 7.65 (s, 1H), 3.23
(s, 2H), 1.48 (s, 9H). 13C NMR (100 MHz, CDCl3) δ: 172.1,
160.8, 154.2, 150.6, 115.4, 82.5, 32.4, 27.9. HRMS (EI)
calcd. for C11H15N3O5: 269.1012 [M+]; found: 269.1015.
Acknowledgements
Benzyl N-[2-(fluorenylmethoxycarbonyl)aminoethyl]-N-
[(2-N-(tert-butoxycarbonyl)isocytosin-5-
We thank the Natural Science and Engineering Research
Council of Canada (NSERC) for funding this work through
their Discovery Grants program. FW acknowledges an
NSERC PGS-D scholarship. Mr. Doug Hairsine, Department
of Chemistry, is thanked for performing HRMS of small
molecules. Ms. Paula Pittock of the Biological Mass Spec-
trometry Laboratories (UWO) is thanked for characterization
of the polyamide.
yl)acetyl]glycinate (5)
To an ice-cooled suspension of 4 (400 mg, 1.5 mmol) and
benzyl N-[2-(Fmoc)aminoethyl]glycinate (430 mg, 1.0 mmol)
in DMF (10 mL) was added EDC (575 mg, 3.0 mmol), and
the reaction was stirred on ice for 0.5 h and at room temper-
ature for 36 h. The reaction was poured into NaHCO3
(50 mL) and extracted into CH2Cl2 (3 × 100 mL). The or-
ganic phase was washed sequentially with aqueous NaHCO3
(2 × 50 mL) and brine (50 mL). After drying the organic
phase and removal of the solvent, the residue was subjected
to FCC using CH2Cl2/MeOH as the eluent. The combined
fractions were reduced in vacuo to give 5 (523 mg, 74%). 1H
NMR (400 MHz, DMSO-d6) δ: 11.44 (br s, 1H), 11.01 (br s,
1H), 7.88 (d, J = 7.2 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H),
7.43–7.29 (m, 10H), 5.17 (s, 0.6 H, mi.), 5.12 (s, 1.4 H,
ma.), 4.41–4.19 (m, 4H), 4.08 (s, 1H), 3.51–3.07 (m, 6H),
1.47 (s, 9 H). 13C NMR (100 MHz, CDCl3) δ: 171.3, 170.5,
169.5, 160.4, 156.4, 153.1, 152.0, 150.9, 143.6, 140.9,
135.0, 128.4, 128.3, 128.1, 127.9, 127.3, 126.8, 124.9,
119.7, 115.8, 84.2, 67.2, 66.8, 66.5, 51.2, 49.3, 48.6, 47.0,
39.3, 30.2, 27.8. HRMS (ESI-TOF) calcd. for C37H39N5O8:
704.2696 [MNa+]; found: 704.2687.
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© 2008 NRC Canada