Peptide nucleic acid monomers: A convenient and efficient synthetic approach to Fmoc/Boc monomers

Article abstract of DOI:10.1071/CH11471

A convenient and cost-effective method for the synthesis of Fmoc/Boc-protected peptide nucleic acid monomers is described. The Fmoc/Boc strategy was developed in order to eliminate the solubility issues during peptide nucleic acid solid-phase synthesis, in particular that of the cytosine monomer, that occurred when using the commercialized Bhoc chemistry approach.

Full text of DOI:10.1071/CH11471

CSIRO PUBLISHING
Aust. J. Chem. 2012, 65, 539–544
Full Paper
http://dx.doi.org/10.1071/CH11471
Peptide Nucleic Acid Monomers: A Convenient and
Efficient Synthetic Approach to Fmoc/Boc Monomers
A
B
A C
,
Elisse C. Browne, Steven J. Langford, and Belinda M. Abbott
A
La Trobe Institute of Molecular Science and Department of Chemistry,
La Trobe University, Bundoora, Vic. 3086, Australia.
B
School of Chemistry, Monash University, Clayton, Vic. 3800, Australia.
C
Corresponding author. Email: b.abbott@latrobe.edu.au
A convenient and cost-effective method for the synthesis of Fmoc/Boc-protected peptide nucleic acid monomers is
described. The Fmoc/Boc strategy was developed in order to eliminate the solubility issues during peptide nucleic acid
solid-phase synthesis, in particular that of the cytosine monomer, that occurred when using the commercialized Bhoc
chemistry approach.
Manuscript received: 12 December 2011.
Manuscript accepted: 10 February 2012.
Published online: 14 May 2012.
Introduction
in correspondingly poor yields and difficulties in handling the
purified material. The PNA monomers also require great care in
storage and use to avoid degradation or the absorption of
moisture, again resulting in a significant decrease in reaction
yield. Particular difficulties are also encountered when solubi-
lizing the monomers in the carrier solvent of N-methyl-2-
pyrrolidone (NMP) for automated synthesis, particularly for
the cytosine monomer, which appears to be the most problem-
atic. This results in large amounts of time spent preparing the
monomer solutions using agitation, with sonication also often
required. Clearing the lengthy delivery lines of the automated
PNA synthesizer, if later precipitation occurs, is also very time-
consuming and inconvenient.
Peptide nucleic acid (PNA) is an unnatural oligonucleotide
polymer analogous to the biopolymers deoxyribonucleic acid
and ribonucleic acid, which carry and encode genetic informa-
tion. PNA consists of a backbone of repeating N-(2-aminoethyl)
glycine units connected by peptide bonds with methylene car-
bonyl linkers attaching the traditional nucleotide bases: adenine
(A), guanine (G), cytosine (C), thymine (T) or uridine (U).[1]
Advantages of PNA chemistry include the ability to form an
achiral polymer with unnatural amino acid monomers that are
less susceptible to proteases, have a greater pH stability and are
amenable to peptide–base coupling techniques.[2–3]
A considerable amount of interest has developed around
PNA and similar oligonucleotides owing to their potential appli-
cations in diagnostics, biosensors and antisense therapy.[1,3,4]
Our work has focussed on the development of PNA sequences
that have shown efficacy in a transgenic mouse model
for amyotrophic lateral sclerosis, a form of motor neurone
disease.[5–6] Synthesis of the PNA of interest has required
significant amounts of the corresponding monomers. These
building blocks are commercially available, with fluorenyl-
methyloxycarbonyl (Fmoc) protection of the N-(2-aminoethyl)
glycine backbone and, for the adenine, guanine and cytosine
monomers, benzhydryloxycarbonyl (Bhoc)-protection of the
exocyclic amines of the nucleobase. The desired PNA molecules
are synthesized on resin by automated techniques, isolated
manually, purified by reverse-phase (RP)-HPLC and typically
characterized by matrix-assisted laser desorption–ionization
time-of-flight (MALDI-TOF) mass spectrometry. Although this
approach follows standard protocols for PNA synthesis, it is not
without problems. The monomers are available from few
commercial sources, are very expensive (AU$270–308 per gram
of monomer) and can be obtained only in the Bhoc-protected
form. As a consequence of their expense, automated synthesis is
usually undertaken on a very small scale (2 mmol), which results
As a result, we were interested in finding an improved means
of obtaining PNA monomers by developing a convenient
synthetic route using an N-protecting group that would be more
soluble in NMP than the Bhoc group. Although there are some
synthetic methodologies in the literature outlining the synthesis
of monomers with different protecting groups for both the
backbone and base,[7–10] none of the procedures met our needs
very well. In particular, we were interested in the work by
Porcheddu and coworkers in producing a series of N-tert-
butoxycarbonyl (Boc)-protected nucleobases;[11] however, their
synthesis of the N-(-2-Fmoc-aminoethyl)glycine backbone was
temperamental at best. Fmoc protection of ethanolamine was
effective and straightforward; however, significant difficulties
were encountered when attempting to oxidize the alcohol into
the corresponding aldehyde under the conditions described.
Oxidation using stabilized 2-iodoxybenzoic acid (SIBX,
45 wt- % IBX with benzoic acid and isophthalic acid) rather
than the neat IBX as described, as a result of the potential
explosive hazard, appeared to work in moderate yield. However,
we were unable to isolate the corresponding aldehyde with
sufficient purity. These difficulties prompted us to find a better,
more efficient and cleaner solution.
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

540
E. C. Browne, S. J. Langford, and B. M. Abbott
Herein, we describe a convenient synthesis of the four Fmoc-
protected PNA monomers of C, T, G and A with Boc nucleobase
protection as required (Fig. 1).
Thomson et al.[8] The bulky tert-butyl ester was employed to
prevent self-cyclization, which was observed when alkylating
with methyl or ethyl bromoacetate. Hydrolysis of the tert-butyl
ester 4 was easily achieved by acidolysis using trifluoroacetic
acid in dichloromethane in near-quantitative yield. The ethyl
ester was installed by refluxing 5 in ethanol under acidic
conditions to give the ethyl ester backbone 1 in 98 % yield. This
series of reactions is able to be performed in excellent yields,
with compounds obtained easily in very good purity. With an
overall yield of 85 % from an inexpensive and readily available
starting material, we consider this route preferable to those
describing the synthesis of the equivalent methyl ester
backbone.[10,11,15,16]
Synthesis of the four nucleobases was then undertaken in
three to seven steps depending on the protection required owing
to potential side reaction. The synthesis of the thymine PNA
monomer 8 (Scheme 2) was straightforward following the
synthesis of thymine acetic acid 6.[16] Coupling of 6 to the
Fmoc-protected backbone 1 to obtain 7 was achieved in 74 %
yield using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDC). EDC coupling was desired over other
coupling methods such as HBTU/HOBt,[10] N,N0-dicyclohex-
ylcarbodiimide,[16,18] isobutylchloroformate (IBC)[11] and
ByBop[19] as nucleophilic attack of the secondary amine easily
displaces the O-acylisourea intermediate and the resulting urea
by-product is water-soluble. In addition to this, EDC is safer to
handle and the coupling conditions used herein did not require
the addition of HOBt as described in other coupling proce-
dures.[9,13] Base hydrolysis of the ethyl ester was best achieved
using 1 M LiOH in THF at 08C. The use of 2.5 M NaOH[11] led to
Fmoc cleavage at a faster rate than that of the ethyl ester.
Attempted optimization of NaOH concentration, reaction time
and temperature were unsuccessful. Using LiOH also led to
cleavage of the Fmoc group, but to a lesser extent. To overcome
this issue, Fmoc N-hydroxysuccinimide ester was added to the
reaction mixture to reprotect the cleaved amine to give the
completed thymine PNA monomer 8.[20]
Results and Discussion
Our approach uses an efficient route centred on the production
of an ethyl ester monomer 1. It was originally envisaged that a
tert-butyl ester would be critical for efficiency of preparation, as
it was suggested in the literature that this functionality would be
selectively cleaved by zinc bromide while retaining N-Boc
protection.[12] Unfortunately, this desired orthogonal cleavage
was not observed. Indeed, HPLC monitoring of the reaction
showed that N-Boc deprotection was occurring preferentially.
Therefore, the ethyl ester was determined to be a more suitable
candidate for the protection of the carboxylic acid group. The
ethyl ester was desired as cleavage of benzyl or allyl esters, such
as those used by Hudson et al.,[9,13] require expensive and toxic
reagents such as Pd/C and Pd(PPh3)4 whereas LiOH could be
used for the ethyl ester hydrolysis. This is particularly beneficial
when undertaking synthesis on a larger scale.
The Fmoc protected N-(2-aminoethyl)glycine backbone was
prepared as shown in Scheme 1. Although this compound has
been claimed in the patent literature,[14] no synthetic methodol-
ogy or characterization data could be found. In our method,
ethylenediamine was alkylated with one equivalent of tert-butyl
bromoacetate in approximately 92 % yield, followed by Fmoc
protection of the primary amine in 95 % yield to give the
tert-butyl N-(2-(N-9-fluorenylmethoxycarbonyl)aminoethyl)
glycinate hydrochloride salt 4, according to the procedure by
B
B
B
O
O
O
O
O
O
N
N
N
N
H
N
H
H2N
OH
n
Synthesis of the remaining three nucleobase monomers
required protection of the exocyclic amines in order to improve
Fig. 1. Peptide nucleic acid where B ¼ A, G, C, T or U.
O
O
O
NH
Br2CH2COOtBu
H
1, EDC
DMF, 0ꢀC
NH2
N
NH
H2N
H2N
O
N
O
O
CH2Cl2
3
2
N
O
O
Fmoc-OSu
DIPEA
CH2Cl2
N
COOH
Fmoc
N
H
O
6
7
O
O
O
H
N
H
N
1. LiOH, THF, 0ꢀC
Fmoc
TFA
CH2Cl2
N
H
Fmoc
N
H
OH
HCl
2. Fmoc-OSu
HCl
4
5
O
EtOH
H2SO4
NH
O
N
Mol sieves
O
O
O
H
N
N
Fmoc
N
H
OH
Fmoc
N
H
O
HCl
8
1
Scheme 1. Synthesis of the Fmoc-protected ethyl ester backbone.
Scheme 2. Synthesis of the thymine peptide nucleic acid monomer.

Peptide Nucleic Acid Monomers: A Synthetic Approach
541
coupling efficiency and hence prevent side reactions. The acid-
labile protecting group Boc was installed over the classical Bhoc
group in order to improve the solubility of the PNA monomers in
NMP, particularly the cytosine derivative. Synthesis of the Boc-
protected cytosine monomer is shown in Scheme 3. Preparation
of the cytosine acetic acid 13 was achieved by Boc protection of
9, followed by selective deprotection, alkylation and saponifi-
cation.[11] The N-(2-aminoethyl)glycinate ethyl ester backbone
1 was coupled to the protected cytosine acetic acid derivative 13
using EDC in 71 % yield. This was preferred over the IBC,[11] as
the IBC reaction was not high-yielding and led to several
undesired side products. Saponification of 14 with LiOH and
subsequent reprotection with Fmoc N-hydroxysuccinimide ester
afforded the completed cytosine PNA monomer 15. The
solubility of the Boc-protected cytosine monomer 15 was
determined to be more than six times that of the corresponding
Bhoc-protected monomer in NMP. Furthermore, 15 did not
precipitate out of solution after several days.
The synthesis of the final two Boc-protected purine mono-
mers is shown in Scheme 4. Preparation of the adenine nucleo-
base 16 and the more complex guanine analogue 19 followed the
methodology of Porcheddu.[11] 16 and 19 were coupled with the
Fmoc-protected N-(2-aminoethyl)glycine backbone 1, follow-
ing activation with EDC, in 73 and 69 % yield respectively.
Deprotection of 17 and 20 was undertaken using LiOH and
subsequent reprotection with Fmoc N-hydroxysuccinimide ester
resulted in the completed nucleobases 18 and 21 in high yields
(81 and 89 % respectively).
Boc
Boc
N
N
NH2
Boc2O,
N
DMAP, THF
N
O
N
H
O
Boc
9
10
NaHCO3
MeOH
Conclusion
Boc
Boc
N
Boc
Boc
N
N
A practical and effective synthetic route to the preparation of
Fmoc/Boc protected PNA monomers from inexpensive starting
materials has been reported. This is a much easier, more con-
venient method compared with those currently available. The
synthesis and characterization of N-(2-(Fmoc)aminoethyl)
glycinate ethyl ester and the subsequent four novel monomer
ethyl esters have not been reported in the literature to date.
The coupling of the nucleobases to the backbone is simple
and efficient using EDC. The novel ethyl esters are hydrolysed
in mild basic conditions although reprotection of the Fmoc
backbone is required as a fraction of the protecting group is
cleaved, contrary to other reports in the literature. Most impor-
tantly, the introduction of Boc protection has increased the
solubility of the cytosine building block over six-fold without
crystallization over time.
N
NaH, BrCH2COOMe
DMAP, THF
N
O
O
N
H
COOR
11
12 R ꢁ Me
13 R ꢁ H
NaOH
THF/H2O
1, EDC, DMF
0ꢀC
Boc
N
Boc
N
Boc
Boc
N
N
N
1. LiOH, THF, 0
2. Fmoc-OSu
ꢀC
N
O
O
O
O
Experimental
O
O
N
N
NMR spectra were recorded on a Bruker Avance-300 spec-
trometer (1H at 300.13 MHz and 13C at 75.47 MHz). TLC was
performed using Merck Kieselgel 60 F254 plates. Drying and
purification methods for solvents and reagents were followed
from procedures described by Armarego and Chai.[21] Melting
Fmoc
Fmoc
O
N
H
N
H
OH
14
15
Scheme 3. Synthesis of the cytosine peptide nucleic acid monomer.
Boc
N
Boc
N
Boc
Boc
N
N
Boc
Boc
N
N
N
N
N
N
N
N
N
O
N
O
1, EDC, DMF
1. LiOH, THF, 0ꢀC
O
O
N
O
2. Fmoc-OSu
Fmoc
N
Fmoc
N
N
H
COOH
N
OH
H
16
17
18
O
O
O
N
N
N
N
NH
N
NH
N
N
Boc
NH
Boc
N
1, EDC, DMF
N
O
N
O
N
1. LiOH, THF, 0ꢀC
Boc
Boc
N
Boc
O
2. Fmoc-OSu
O
Boc
Fmoc
N
COOH
Fmoc
N
N
H
O
N
OH
H
19
20
21
Scheme 4. Synthesis of the adenine and guanine peptide nucleic acid monomers.

542
E. C. Browne, S. J. Langford, and B. M. Abbott
points were measured on a hot-stage Reichert Thermopan
apparatus and are uncorrected. Low-resolution electrospray
ionization (ESI) mass spectrometry was carried out using an
Esquire 6000 ion-trap mass spectrometer (Bruker Daltonics,
Germany). The samples were introduced at a flow rate of
4 mL minꢀ1 and a mass range of 50–3000 m/z was recorded. A
scan rate of 5500 m/z sꢀ1 was used with the temperature set at
3008C. High-resolution electrospray ionization mass spec-
trometry (HR-ESI) was performed on an Agilent Technologies
6220 accurate-mass TOF (time of flight) LC/MS as the solutions
specified. [M]þ denotes the molecular ion. Analyses were per-
formed in positive ion mode (ESIþ) unless otherwise stated.
1.70 (s, 18H, 3 ꢁ CH3), 1.22–1.12 (m, 3H, CH2–CH3). dC
(75 MHz, [D6]DMSO) 169.4, 167.8, 164.8, 156.8, 151.4, 144.3,
142.5, 141.2, 128.1, 127.5, 125.6, 120.6, 65.9, 61.0, 48.4, 48.6,
47.3, 47.2, 14.5, 12.4. m/z (HR-ESI) 535.2196 (M þ H),
557.2014 (M þ Na).
2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetamido)acetic Acid 8
Compound 7 (173 mg, 0.32 mmol) was dissolved in THF (2 mL)
and chilled to 08C. To this, 2 equiv. of LiOH (1 M) was added
dropwise and the reaction was stirred for 30 min, followed by the
addition of Fmoc-succinimide (109 mg, 0.32 mmol), and
the reaction stirred for 12 h. The resultant solid was filtered and
the residue dissolved in hot EtOAc and precipitated with hexane
to give 8 as a white solid (163 mg, 85 %), mp 220–2228C (lit.[4]
216–2198C). dH (300 MHz, [D6]DMSO) 11.23 (s, 1H, OH), 7.84
(d, J 7.2, 2H, 2 ꢁ CH), 7.64 (d, J 7.2, 2H, 2 ꢁ CH), 7.48 (bt, 1H),
7.38 (t, J 7.2, 2H, 2 ꢁ CH), 7.30 (t, J 7.2, 2H, 2 ꢁ CH). dC
(75 MHz, [D6]DMSO) 171.4, 167.8, 164.5, 156.2, 151.1, 144.0,
142.28, 140.8, 127.7, 127.2, 125.4, 120.2, 108.0, 65.6, 51.7,
47.8, 47.5, 46.9, 46.8, 12.0. m/z (LR-ESI) 507.4 (M þ H), 529.4
(M þ Na).
Ethyl 2-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)amino)acetate Hydrochloride Salt 1
A solution of 5 (3 g, 7.96 mmol) in dry ethanol (30 mL) and
˚
H2SO4 (500 mL) was left to reflux over 3-A molecular sieves
overnight. The hot solution was filtered and concentrated, and
the addition of cold 1 M HCl precipitated 1 as a white solid
(3.15 g, 98 %), mp 150–1518C. dH (300 MHz, [D6]DMSO) 9.52
(s, 2H, NH), 7.85 (d, J 7.5, 2H, 2 ꢁ CH), 7.66 (d, J 7.5, 2H,
2 ꢁ CH), 7.53 (t, J 5.4, 1H), 7.38 (t, J 7.5, 2H, 2 ꢁ CH), 7.30 (t, J
7.5, 2H, 2 ꢁ CH), 4.29 (d, J 6.3, 2H, CHCH2), 4.20–4.131 (m,
4H, CH2CH3 and CHCH2), 3.93 (s, 2H, CH2NH), 3.30 (m, 2H,
CH2CH2), 2.99 (t, J 6.0), 1.20 (t, J 7.2, 3H, CH2CH3). dC
(75 MHz, [D6]DMSO) 167.0, 156.7, 144.3, 144.3, 141.2, 128.1,
127.5, 125.6, 120.6, 66.1, 62.1, 47.1, 14.9, 37.0, 14.4. m/z
(HR-ESI) 369.1816 (M þ H), 391.1635 (M þ Na).
Ethyl 2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(4-(bis(tert-butoxycarbonyl)amino)-2-
oxopyrimidin-1(2H)-yl)acetamido)acetate 14
A solution of 13 (400 mg, 1.08 mmol) in anhydrous DMF was
cooled to 08C under a blanket of argon. To this, EDC (372 mg,
7.94 mmol) was added and the reaction stirred for 30 min, fol-
lowed by the addition of 1 (460 mg, 1.13 mmol), and the reaction
left to stir overnight. The reaction was quenched on ice, the
resulting solid filtered, purified by column chromatography
(7 : 3 EtOAc : hexane) and trituated with diethyl ether to give 14
as white solid (550 mg, 71 %), mp 81–838C. dH (300 MHz, [D6]
DMSO, two rotamers 3 : 2) 7.98–7.92 (m, 1H, rotamer vinylic
H), 7.85 (d, J 7.5, 2H), 7.65 (d, J 7.2, 2H), 7.34–7.23 (m, 5H,
4 ꢁ Ar–CH, NH), 6.80–6.76 (m, 1H, rotamer vinylic H), 4.83 (s,
12.H, N–CH2–C¼O), 4.65 (s, 0.8H, N–CH2–C¼O), 4.32–4.01
(m, 7H, CH2–CH3, CH–CH2, N–CH2–C¼O), 3.43–3.06 (m, 4H,
CH2–CH2), 1.70 (s, 18H, 3 ꢁ CH3), 1.23–1.12 (m, 3H, CH2–
CH3). dC (75 MHz, [D6]DMSO) 169.4, 167.5, 162.4, 156.8,
154.5, 152.0, 149.7, 144.3, 141.2, 128.0, 127.5, 125.6, 120.6,
95.7, 85.0, 66.0, 61.0, 50.2, 48.4, 47.5, 47.1, 27.7, 14.5. m/z
(HR-ESI) 720.3245 (M þ H), 742.3061 (M þ Na).
2-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethyl)
amino)acetic Acid Hydrochloride Salt 5
A solution of 4 (3.0 g, 6.93 mmol) in a 1 : 1 solution of TFA/
CH2Cl2 (20 mL) was stirred for 1 h. Toluene (20 mL) was added
to the solution and the solvents evaporated under vacuum. This
process was repeated twice. NaHCO3 was added to the residue,
followed by acidification using 6 M HCl until precipitation
ceased. The product was filtered by vacuum filtration, washed
with water and further dried in a desiccator overnight to give 5 as
a white solid (2.48 g, 98 %), mp 156–1598C (lit.[22] 154–1568C).
dH (300 MHz, [D6]DMSO) 9.32 (bs, 1H, OH), 7.86 (d, J 7.5, 2H,
2 ꢁ CH), 7.65 (d, J 7.2, 2H, 2 ꢁ CH), 7.48 (bt, 1H), 7.38 (t, J 7.2,
2H, 2 ꢁ CH), 7.30 (t, J 7.2, 2H, 2 ꢁ CH), 4.30 (d, J 6.9, H,
CHCH2), 4.19 (t, J 6.6, 1H, CHCH2), 3.29 (m, 2H, CH2CH2),
2.98 (t, J 6.0, 2H, CH2CH2). dC (75 MHz, [D6]DMSO) 183.8,
168.4, 144.3, 141.2, 128.1, 127.5, 125.6, 120.6, 66.07, 47.4,
47.1, 46.9, 37.1.
2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(4-(bis(tert-butoxycarbonyl)amino)-2-
oxopyrimidin-1(2H)-yl)acetamido)acetic Acid 15
Ethyl 2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetamido)acetate 7
Compound 14 (150 mg, 0.20 mmol) was dissolved in THF
(2 mL) and chilled to 08C. To this, 2 equiv. of LiOH (1 M) was
added dropwise and the reaction was stirred for 30 min, followed
by the addition of Fmoc-succinimide (70 mg, 0.20 mmol), and
the reaction stirred for 12 h. The resultant solid was filtered and
recrystallized by ethyl acetate and hexane to give 15 as a white
solid (120 mg, 83 %), mp 115–1168C (lit.[5] 110–1128C). dH
(300 MHz, [D6]DMSO, two rotamers 3 : 2) 7.97–7.92 (m, 1H,
rotamer vinylic H), 7.86 (d, J 7.2, 2H), 7.65 (d, J 7.2, 2H), 7.40–
7.22 (m, 5H, 4 ꢁ Ar–CH, NH), 6.80–6.74 (m, 1H, rotamer
vinylic H), 4.83 (s, 12.H, N–CH2–C¼O), 4.65 (s, 0.8H, N–CH2–
C¼O), 4.32–4.20 (m, 3.8H, CH–CH2, N–CH2–C¼O, 0.8H),
3.97 (s, 1.2H, CH2), 3.42–3.09 (m, 4H, CH2–CH2), 1.45 (s, 18H,
To a solution of 6 (275 mg, 1.49 mmol) in anhydrous DMF
(5 mL) at 08C under argon, EDC (514 mg, 2.68 mmol) was
added and stirred for 30 min. To this, 1 (635 mg, 1.57 mmol) was
added portionwise over a period of 30 min and the reaction
stirred overnight. The reaction mixture was poured over ice and
the resultant solid was filtered off and purified by flash chro-
matography (EtOAc) to give 7 as a white solid (583 mg, 74 %),
mp 160–1628C. dH (300 MHz, [D6]DMSO, two rotamers 3 : 2)
11.22 (s, NH), 7.85 (d, J 7.5, 2H), 7.65 (d, J 7.5, 2H), 7.40–7.22
(m, 6H, 4 ꢁ Ar–CH, NH, vinylic H), 4.63 (s, 1.2H, N–CH2–
C¼O), 4.45 (s, 0.8H, N–CH2–C¼O), 4.32–3.98 (m, 7H, CH2–
CH3, CH–CH2, N–CH2–C¼O), 3.42–3.04 (m, 4H, CH2–CH2),

Peptide Nucleic Acid Monomers: A Synthetic Approach
543
3 ꢁ CH3). dC (75 MHz, [D6]DMSO) 170.8, 167.3, 162.4, 156.8,
154.6, 152.0, 149.7, 144.4, 141.2, 128.1, 127.5, 125.6, 120.6,
95.7, 85.0, 66.0, 50.2, 48.2, 47.4, 47.2, 27.7. m/z (LR-ESI) 692.3
(M þ H), 714.3 (M þ Na).
7.65 (d, J 7.2, 2H, 2 ꢁ CH), 7.37 (t, J 7.5, 2H, 2 ꢁ CH), 7.28 (t,
J 7.5, 2H, 2 ꢁ CH), 5.18 (s, 1.2H, NCH2C¼O), 5.18 (s, 0.8H,
NCH2C¼O), 4.37–3.96 (complex m, 7H), 3.50 (br t, 2H), 3.10
(br t, 2H), 1.31 (s, 18H, 2 ꢁ C(CH3)3), 1.13 (t, J 7.2, 3H,
CH2CH3). dC (75 MHz, [D6]DMSO) 169.3, 167.2, 156.8, 156.6,
154.9, 152.1, 151.4, 144.3, 141.8, 141.2, 128.1, 127.5, 125.5,
120.6, 117.6, 82.5, 65.9, 61.0, 49.6, 48.4, 47.4, 44.0, 27.8, 14.4.
m/z (HR-ESI) 760.3303 (M þ H), 782.3120 (M þ Na).
Ethyl 2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)
acetamido)acetate 17
A solution of 16 (450 mg, 1.14 mmol) in anhydrous DMF (5 mL)
was cooled to 08C under a blanket of argon. To this, EDC
(395 mg, 2.06 mmol) was added and the reaction stirred for
30 min, followed by the addition of 1 (508 mg, 1.25 mmol), and
the reaction left to stir overnight. The reaction was quenched on
ice, the resulting pink solid filtered, purified by column chro-
matography (4 : 1 EtOAc : hexane) and coevaporated with die-
thyl ether to give 17 as white solid (617 mg, 73 %), mp 85–888C.
dH (300 MHz, [D6]DMSO, two rotamers 3 : 2) 8.76 (s, 0.3H,
minor vinylic rotamer), 8.44 (s, 0.7H, major vinylic rotamer),
8.44 (d, 1H, vinylic rotamer), 7.86–7.82 (m, 2H, 2 ꢁ Ar–H),
7.66–7.62 (m, 2H, 2 ꢁ Ar–H), 7.46–7.24 (m, 5H, 2 ꢁ Ar–H,
NH), 5.37 (s, 1.2H, N–CH2–C¼O), 5.20 (s, 0.8H, N–CH2–
C¼O), 4.41–4.00 (m, 7H, CH2–CH3, CH–CH2, N–CH2–C¼O),
3.54–3.09 (m, 4H, CH2–CH2), 1.34 (s, 18H, 3 ꢁ CH3), 1.24–
1.05 (m, 3H, CH2–CH3). dC (75 MHz, [D6]DMSO) 169.3,
167.0, 156.9, 153.9, 151.9, 150.5, 149.4, 148.1, 144.3, 141.2,
128.1, 127.8, 127.7, 127.5, 125.5, 120.6, 83.7, 66.0, 61.0, 48.4,
47.5, 47.2, 44.9, 44.6, 39.3, 27.7, 14.4. m/z (HR-ESI) 744.3353
(M þ H), 766.3159 (M þ Na).
2-(N-(2((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-
2-(2-(bis(tert-butoxy carbonyl)amino)-6-oxo-1H-purin-
9(6H)-yl-acetamido)acetic Acid 21
Compound 20 (200mg, 0.23mmol) was dissolved in THF (2mL)
and chilled to 08C. To this, 2 equiv. of LiOH (1 M) was added
dropwise and the reaction was stirred for 30min, followed by the
addition of Fmoc-succinimide (13 mg, 0.23mmol), and the
reaction stirred for 12h. The reaction was quenched with 1 M
HCl, extracted with EtOAc (3ꢁ 15mL) and washed with water.
The residue was purified by column chromatography (CH2Cl2 :
MeOH 19: 1) yielding 21 as a white solid (171mg, 89%), mp
2538C (decomp.) (lit.[5] 2558C decomp.). dH (300MHz, [D6]
DMSO, two rotamers 3 : 2) 7.98 (d, 1H, rotamer vinylic H), 7.84–
7.82 (m, 2H, 2 ꢁ Ar–H), 7.49 (bs, 1H, NH), 7.36–7.27 (m, 4H,
4 ꢁ Ar–H), 5.15 (s, 0.8H, N–CH2–C¼O), 4.96 (s, 1.2H, N–CH2–
C¼O), 4.30–3.92 (m, 5H, CH–CH2, N–CH2–C¼O), 3.47–3.12
(m, 4H, CH2–CH2), 1.32 (s, 18H, 3 ꢁ CH3). dC (75 MHz, [D6]
DMSO) 171.9, 167.6, 156.77, 156.54, 154.8, 152.1, 151.4, 144.3,
142.0, 141.2, 128.0, 127.6, 125.7, 120.5, 117.5, 82.5, 66.0, 51.8,
48.1, 48.0, 47.1, 44.2, 27.8. m/z (LR-ESI) 732.2 (Mþ H).
2-(N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)
acetamido)acetic Acid 18
Acknowledgements
We acknowledge funding by the Australian Research Council (DP0771578).
E.C.B. is also the recipient of an Australian Postgraduate Award.
Compound 17 (200 mg, 0.27 mmol) was dissolved in THF
(2 mL) and chilled to 08C. To this, 2 equiv. of LiOH (1 M) was
added dropwise and the reaction was stirred for 30 min, followed
by the addition of Fmoc-succinimide (90 mg, 0.27 mmol), and
the reaction stirred for 12 h. The resultant solid was filtered and
recrystallized by ethyl acetate and hexane to give 18 as a white
solid (156 mg, 81 %), mp 105–1068C (lit.[5] 103–1048C). dH
([D6]DMSO, two rotamers 3 : 2) 9.98 (s, 1H, OH), 8.75 (s, 0.4H,
CH), 8.70 (s, 0.6H, CH), 8.44 (s, 1H, CH), 7.90–7.81 (m, 2H,
2 ꢁ CH), 7.67–7.58 (m, 2H, 2 ꢁ CH), 7.50–7.35 (m, 5H, 4 ꢁ CH,
NH), 5.37 (s, 1.2H, NCH2C¼O), 5.19 (s, 0.8H, NCH2C¼O),
4.34–4.20 (complex m, 5H), 3.98 (s, 2H, CH2), 3.53–3.12 (m,
4H), 1.34 (s, 18H, 2 ꢁ C(CH3)3). dC (75 MHz, [D6]DMSO)
173.2, 170.8, 166.8, 156.9, 153.9, 151.5, 150.2, 149.4, 148.3,
144.3, 141.2, 141.1, 128.1, 127.8, 127.5, 125.6, 125.5, 120.6,
83.7, 66.0, 49.8, 48.1, 47.4, 47.2, 44.8, 38.4, 27.7. m/z (LR-ESI)
716.6 (M þ H), 738.5 (M þ Na).
References
[1] P. E. Nielsen, Science 1991, 254, 1497. doi:10.1126/SCIENCE.
1962210
[2] K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen,
T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt,
J. Org. Chem. 1994, 59, 5767. doi:10.1021/JO00098A042
[3] P. E. Nielsen, M. Egholm, O. Buchardt, Bioconjug. Chem. 1994, 5, 3.
doi:10.1021/BC00025A001
[4] A. Ray, B. Norden, FASEB J. 2000, 14, 1041.
[5] I. K. Cheah, S. S. Cheema, S. J. Langford, E. C. Lopes,
K. J. Macfarlane, S. Petratos, B. J. Turner, Bioorg. Med. Chem. Lett.
2003, 13, 2377. doi:10.1016/S0960-894X(03)00400-1
[6] B. J. Turner, I. K. Cheah, K. J. Macfarlane, E. C. Lopes, S. Petratos,
S. J. Langford, S. S. Cheema, J. Neurochem. 2003, 87, 752.
doi:10.1046/J.1471-4159.2003.02053.X
[7] S. Pothukanuri, Z. Pianowski, N. Winssinger, Eur. J. Org. Chem. 2008,
3141. doi:10.1002/EJOC.200800141
Ethyl 2-(N-(2(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)
ethyl)-2-(2-(bis(tert-butoxy carbonyl)amino)-6-oxo-1H-
purin-9(6H)-yl-acetamido)acetate 20
[8] S. A. Thomson, J. A. Josey, R. Cadilla, M. D. Gaul, C. Fred Hassman,
M. J. Luzzio, A. J. Pipe, K. L. Reed, D. J. Ricca, R. W. Wiethe,
S. A. Noble, Tetrahedron 1995, 51, 6179. doi:10.1016/0040-4020(95)
00286-H
[9] F. Wojciechowski, R. H. E. Hudson, J. Org. Chem. 2008, 73, 3807.
doi:10.1021/JO800195J
To a solution of 19 (912 mg, 2.23 mmol) and 1 (890 mg,
2.23 mmol) in anhydrous DMF (10 mL), EDC (855 mg,
4.45 mmol) was added portionwise over a period of 1 h and
stirred under argon overnight. The reaction mixture was poured
over ice and the resultant solid was filtered off. The resulting
solid was purified by column chromatography (4 : 1 EtOAc :
hexane) and trituated with diethyl ether to give 20 as a white
crystalline solid (1.16 g, 69 %), mp 87–898C. dH ([D6]DMSO,
two rotamers 3 : 2) 7.98 (s, 1H, NCH), 7.84 (d, J 7.5, 2H, 2 ꢁ CH),
[10] T. Stafforst, U. Diederichsen, Eur. J. Org. Chem. 2007, 681.
doi:10.1002/EJOC.200600747
[11] A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta, G. Nieddu, Eur.
J. Org. Chem. 2008, 5786. doi:10.1002/EJOC.200800891
[12] Y.-q. Wu, D. C. Limburg, D. E. Wilkinson, M. J. Vaal, G. S. Hamilton,
Tetrahedron Lett. 2000, 41, 2847. doi:10.1016/S0040-4039(00)
00300-2

544
E. C. Browne, S. J. Langford, and B. M. Abbott
[13] A. H. St. Amant, R. H. E. Hudson, Org. Biomol. Chem. 2012, 10, 876.
doi:10.1039/C1OB06582C
[14] J. M. Coull, M. Egholm, R. P. Hodge, M. Ismail, S. B. Rajur, PCT
[18] O. Seitz, O. Ko¨hler, Chemistry 2001, 7, 3911. doi:10.1002/1521-3765
(20010917)7:18,3911::AID-CHEM3911.3.0.CO;2-1
[19] D. V. Jarikote, O. Ko¨hler, E. Socher, O. Seitz, Eur. J. Org. Chem. 2005,
15, 3187. doi:10.1002/EJOC.200500201
Patent WO 96/40685, 1996.
[15] L. Bialy, J. J. D´ıaz-Mocho´n, E. Specker, L. Keinicke, M. Bradley,
Tetrahedron 2005, 61, 8295. doi:10.1016/J.TET.2005.06.003
[16] R. H. E. Hudson, Y. Liu, F. Wojciechowski, Can. J. Chem. 2007, 85,
302. doi:10.1139/V07-030
[20] S. Chauhan, A. Varshney, B. Verma, M. Pennington, Tetrahedron Lett.
2007, 48, 4051. doi:10.1016/J.TETLET.2007.04.027
[21] W. L. F. Armarego, C. L. L. Chai, in Purification of Laboratory
Chemicals (5th edn) 2003 (Elsevier: Cornwall).
[17] X.-J. Liu, R.-Y. Chen, Phosphorus Sulfur 2001, 176, 19. doi:10.1080/
10426500108055099
[22] D. Milic, M. Prato, Eur. J. Org. Chem. 2010, 476. doi:10.1002/EJOC.
200900791