A practical and efficient approach to PNA monomers compatible with Fmoc-mediated solid-phase synthesis protocols

Article abstract of DOI:10.1002/ejoc.200800891

A straightforward synthesis of orthogonally protected PNA monomers is described. Protected aminoethylglycine (Aeg) monomers were efficiently prepared by reductive amination of N-Fmoc-glycinaldehyde with glycine methyl ester and the subsequent acylation of

Full text of DOI:10.1002/ejoc.200800891

FULL PAPER
DOI: 10.1002/ejoc.200800891
A Practical and Efficient Approach to PNA Monomers Compatible with
Fmoc-Mediated Solid-Phase Synthesis Protocols
Andrea Porcheddu,*[a] Giampaolo Giacomelli,[a] Ivana Piredda,[a] Mariolino Carta,[a] and
Giammario Nieddu[a]
Dedicated to the memory of Dr. Charles Mioskowski
Keywords: Peptide nucleic acids / Guanine / Protecting groups / Solid-phase synthesis
A straightforward synthesis of orthogonally protected PNA
monomers is described. Protected aminoethylglycine (Aeg)
monomers were efficiently prepared by reductive amination
of N-Fmoc-glycinaldehyde with glycine methyl ester and the
group; this increased the solubility of the nucleobases in the
most common organic solvents. The current protocol allows
all Aeg monomers to be prepared on both the micro- and
macroscale, which avoids or minimizes the use of toxic rea-
subsequent acylation of the free amine with N-bis-Boc-pro- gents or solvents, and moreover, cheap starting materials are
tected nucleobase acetic acids. The exocyclic amine group of
the nucleobases, including the notoriously difficult-to-protect
guanine nucleobase, was protected with a bis-Boc carbamate
used.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
same time, the change in ionic strength has little effect on
the stability of PNA–DNA duplexes.[3] They also possess
high chemical and biological stability due to the unnatural
amide backbone that is not recognized by nucleases or pro-
teases and thus not degraded inside a living cell.[4] These
properties of PNAs make these oligomers of significant
interest in many disciplines of chemistry, molecular biology
and medicine.[5] A ubiquitous requirement in the field of
PNAs research[6] is the preparation of monomers for subse-
quent oligomerization. A PNA monomer consists of N-pro-
tected (2-aminoethyl)glycine to which a protected nucleo-
base is attached (Figure 1). These two protecting groups
have to be orthogonal, that is, Pg2 must be stable under the
conditions used to remove Pg1.
Introduction
Natural oligonucleotides (DNA, RNA) have limited po-
tential as therapeutic agents, and main reason is their poor
in vivo stability as a result of the susceptibility of the phos-
phodiester backbone to undergo enzymatic cleavage by
cellular nucleases. To overcome this general problem in the
antisense field, there have been significant advances in the
discovery and development of analogues bearing structural
features that improve the pharmacological properties of
natural oligonucleotides. Peptide nucleic acids (PNAs) are
DNA/RNA mimics in which the sugar–phosphate back-
bone of natural nucleic acid has been replaced by an un-
charged pseudopeptide skeleton composed of N-(2-aminoe-
thyl)glycine units (Figure 1).[1] The nucleobases A, C, G and
T are linked to this achiral skeleton through a two-atom
carboxymethyl spacer. PNAs have very flexible structures,
but they are still able to bind complementary DNA and
RNA strands with high specificity and selectivity.[2] PNA–
RNA and PNA–DNA hybrids are more stable than the cor-
responding nucleic acid complexes. The increased stability
of PNA–DNA and PNA–RNA duplex in comparison to
DNA–DNA (RNA) duplex is mainly attributed to the lack
of electrostatic repulsion between the two strands. At the
Figure 1. General structures of a PNA monomer.
In the literature, during these last years several combina-
tions of protecting groups were reported for PNA synthe-
sis.[7] PNA oligomers can be prepared by following standard
solid-phase synthesis protocols for peptides by using, for
example, a (methylbenzhydryl)amine polystyrene resin as
the solid support.[8] Previous reports on PNA synthesis have
focused on Boc/Cbz protection methods (Pg1 = Boc and
[a] Dipartimento di Chimica, Università degli Studi di Sassari,
Via Vienna 2, 07100 Sassari, Italy
Fax: +39-079-229559
E-mail: anpo@uniss.it
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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A Practical and Efficient Approach to PNA Monomers
Pg2 = Cbz, Figure 1), giving an assortment of synthetic pro-
Such problems may be overcome by using two Boc (bis-
cedures. The repeated use of TFA (Boc group deprotection) Boc) protecting groups, as the complete protection of the
and the harsh treatments (HF or TFMSA) necessary to re- exocylic amine functionality increase the lipophilicity of
move the Cbz protecting group makes this procedure in- bases, which makes them more soluble in the most common
compatible with several modified PNAs, in particular with solvents for organic synthesis. Moreover, it generally known
PNA–DNA chimeras, which are sensitive to strong acids.[9] that the bis-Boc protecting group often avoids side reac-
Moreover, the use of hazardous acids often are not com- tions or unwanted cyclizations.[18] In 1996, Condom et al.
mercially embraced in view of safety concerns for the opera- reported, for the first time, the preparation of a bis-N-Boc-
tors and the corrosive effect on automation equipment and protected adenine PNA acetic acid derivative for the solu-
lines.
tion-phase synthesis of a PNA adenine–guanine dimer.[19]
With the advent of Nielsen’s PNAs and related amide- However, the paper contained only a summary description
linked oligonucleotide surrogates, there is a continuous of experimental conditions, which were disclosed later.[20]
need for protecting groups that can be removed under acidic In 1997, Garner and Dey reported in detail the synthesis of
or neutral conditions that are compatible with 9-fluor- gram quantities of Boc-protected purines from inexpensive
enylmethoxycarbonyl-mediated (Fmoc) solid-phase synthe- starting materials without the need for elaborate purifica-
sis protocols. The use of an Fmoc group for the protection tion steps.[21] In 2006, Hultin et al.[22] synthesized the bis-
of the primary amine in the backbone offers several advan- N-Boc protection of the heterocyclic amino group of nu-
tages, including milder synthesis conditions, improved cleosides and derivatives and showed that this bis-carba-
monomer solubility, high coupling efficiencies and facili- mate is a very useful N-protecting group for the nucleobase
tated purification of the final products.[10]
portions. These last seminal papers encouraged us to de-
Another current limitation on the synthesis of PNA syn- velop a general strategy to prepare Fmoc/bis-N-Boc Aeg–
thons is the formation of the side-chain nucleobase protect- PNA (Aeg = aminoethylglycine) monomers for the follow-
ing group. Although Fmoc/Bhoc PNA monomers[11] are ing oligomerization of PNAs.
nowadays the most used for routine PNA synthesis, they
During the preparation of this manuscript, the beneficial
are costly and of limited variety.[12] Moreover, the employ- effect of the protection of nucleobases with a bis-Boc group
ment of the Bhoc group for protection of the exocyclic in the Fmoc-mediated solid-phase peptide synthesis of
amino groups (Pg2) onto the nucleobases requires toxic rea- PNAs (Fmoc/bis-Boc strategy) was successfully described
gents such as triphosgene, which renders the Fmoc mono- by Hudson and Wojciechowski.[23] At the same time, a pa-
mer synthesis easier said than done.[13]
per on the scope and orthogonality of PNA synthesis was
Although there are several examples of nucleobases with published by Winssinger et al.[24,25]
acid-labile protecting groups,[14] strangely, the tert-butoxy-
carbonyl group (Boc), which is the most popular acid-labile
Results and Discussion
protecting group for amines, has received little attention in
the literature for nucleobase protection.[15] The Boc protect-
The preparation of the monomers can be divided into
ing group has the additional advantage that its acidolytic the synthesis of a suitably protected N-2-aminoethylglycine
removal is less sensitive to steric factors and it can also be (Pg1-Aeg-OPg3) and the nucleobase acetic acid derivatives
removed under neutral conditions.[16] Boc protection was (Scheme 1). In such monomers, the Fmoc group was intro-
envisaged as a highly attractive strategy, as this protecting duced to protect the 2-ethylamino moiety in the growing
group is orthogonal to Fmoc-based solid-phase peptide PNA backbone, whereas the bis-Boc group was chosen as
synthesis (SPPS) protocol, and in contrast to the known the protecting group for the exocyclic amino functions of
acid-labile monomethoxytrityl (Mmt) protecting group, it the A, G and C bases. In the exploration for a useful pro-
can sustain mildly acidic conditions. Thus, with the appro- tecting group (Pg3) for the carboxylic function of Pg1-Aeg-
priate choice of resin/linker, on-resin deprotection and OH, we found methyl ester to be ideal in terms of deactiva-
modification of a specific amine group would also be pos- tion of COOH towards nucleophiles, ease of deprotection
sible.
and compatibility with our Fmoc/Boc strategy. The key in-
In 2002, Sugiyama et al. reported the first example of the termediate in our new synthetic route to PNA building
synthesis of Fmoc PNA monomers, whose nucleobases blocks is N-Fmoc-aminoethylglycine methyl ester
were orthogonally protected with a Boc group.[17] Unfortu- (Scheme 1). We chose the methyl ester,[26] as it has less pro-
nately, in this note the authors do not provide any exhaus- pensity than the allyl ester to give 2-oxopiperazine during
tive experimental procedure or investigate whether the final the carbamylation reaction as observed by Seitz and
monomers are compatible with a solid-phase PNA oligo- Kohler[27] and confirmed by Hudson et al.[28]
mer synthesis. Nonetheless, the N2-Boc protected carboxy-
A very simple and efficient approach to prepare this
methylguanine was found to possess very poor solubility in compound on large scale is shown in Scheme 2. The Fmoc-
common solvents for peptide coupling. Although this prob- protected backbone (Fmoc-Aeg-OH) could be prepared by
lem was overcome by employing a trimethylsily ether pro- reductive amination of N-Fmoc-glycinaldehyde with glycine
tecting group at the guanine O6 position, this last method- methyl ester.
ology is laborious and requires toxic reagents such as OsO4
and NaClO2.
First, we protected the amino group of ethanolamine by
using Fmoc-Cl, following a classical procedure,[29] and then
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procedure to prepare Fmoc-submonomer 5 is straightfor-
ward and inexpensive, no further attempts at optimization
of the reaction yields were taken into consideration. Our
goal was to develop a simple and reproducible procedure
to overcome many of the practical problems observed dur-
ing the synthesis of PNA monomers.
Second, we focused our attention on the synthesis of bis-
Boc protected nucleobase acetic acids by avoiding or mini-
mizing the use of toxic reagents or solvents such as dimeth-
ylformamide[35] (DMF).[36]
Scheme 1. Retrosynthetic scheme for PNA monomers.
The suitably protected nucleobases were prepared ac-
cording to minor modifications of Garner’s procedure,[21]
which allowed these intermediates to be synthesized in
gram quantities by starting from inexpensive starting mate-
rials and without the need for elaborate purification steps.
Cytosine (C) was suspended in dry THF and treated with
an excess amount of Boc2O (4 equiv.) in the presence of a
catalytic amount of 4-N,N-(dimethylamino)pyridine
(DMAP). This mixture was stirred at room temperature un-
til complete solubilization/disappearance of starting nucleo-
base C (overnight). The excess amount of THF was re-
moved under vacuum to give a yellow oil, which was redis-
solved in AcOEt. The organic solvent was first washed with
HCl (1 ) to give an oil containing tris-Boc-protected cyto-
sine 7 and tert-butyl alcohol (Scheme 4). This mixture, after
treatment with hexane, afforded desired and pure com-
pound 7 in quantitative yield. Tris-Boc cytosine 7 can be
easily converted into bis-Boc cytosine 8 almost quantita-
tively by treatment with aq. NaHCO3 in MeOH at reflux
(until disappearance of starting material by TLC). Longer
reaction times led to the formation of significant quantities
of cytosine.
Scheme 2. Synthesis of N-Fmoc-glycinaldehyde.
we oxidized[30] its alcoholic function to the corresponding
aldehyde with IBX.[31] This procedure gave N-Fmoc-pro-
tected glycine 2 (Scheme 2) in high yield and purity. This
material was taken on to the next step without further puri-
fication. Aldehyde 2 was treated with glycine methyl ester
(3) in methanol, and intermediate imine 4 was efficiently
transformed into amine 5 with 10% Pd/C. N-Fmoc pro-
tected monomer backbone 5 was obtained as a pale yellow
oil in 75% yield after purification by flash chromatography
(Scheme 3).
Scheme 3. Synthesis of N-(2-Fmoc-aminoethyl)glycine methyl ester.
One of main drawbacks of N-(2-Fmoc-aminoethyl)gly-
cine methyl ester (5) is that once obtained it can be stored
only for a few days at low temperature.[32] A more attractive
method involved transforming the crude materials into the
corresponding stable hydrochloride salt 6 by treatment of
Scheme 4. Synthesis of bis-Boc citosylacetic acid.
The alkylation of cytosine derivative 8 was performed by
Fmoc-Aeg-OMe (5) with dry methanolic HCl[33] (2 ) at using NaH and methyl bromoacetate in THF to give, after
0 °C, which could be stored at –20 °C indefinitely.[34] Our purification by flash chromatography, methyl cytosin-1-yl-
aim was to either convert this secondary amine (Fmoc-Aeg- acetate 9 in high yield (Scheme 4). Finally, cleavage of the
OMe) into the amide shortly after its synthesis or to isolate methyl ester group under basic conditions (NaOH 1  in
5 as stable salt 6.
THF) gave the corresponding cytosylacetic acid derivative
As known from the literature, and well remarked by 10 as a white solid in high yield (85%, Scheme 4). Different
Hudson et al.,[23] the introduction of the Fmoc group on from the procedure of Hudson, we prefer to introduce the
the primary amine of the 2-aminoethylglycine moiety is protecting group of the nucleobase in the first step, just be-
never trivial and often proceeds in low yield. Because our fore the alkylation step.[37] This simple shrewdness not only
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A Practical and Efficient Approach to PNA Monomers
renders the intermediates sufficiently soluble for chemical
manipulations, but also makes the whole synthesis of PNA
monomers easier.
The next step in the synthesis of the PNA components is
the formation of the N9-acetic acid derivatives of adenine
and guanine. The introduction of bis-N-Boc onto N9-alkyl-
ated adenine was performed by following the protocol al-
ready described for cytosine.[21] Under these conditions, we
obtained bis-N-Boc-protected compound 12 (96%,
Scheme 5).
Figure 2. Bis-Boc-protected guanine monomer.
The development of an easy and successful synthesis of
guanine monomer represents here our more arduous objec-
tive, as the most part of published procedures fail just in
the preparation of this monomer. In contrast, guanine
quadruplexes are gaining increasing attention due to their
suspected roles in regulating gene expression at the tran-
scriptional and translational levels. In a pioneering work,
Armitage et al.[39] demonstrated that PNAs are capable of
forming a hybrid quadruplex with DNA, a structure com-
posed of PNA2–DNA2. PNA2–DNA2 quadruplex may po-
tentially act as a repressor in diverse gene expression regula-
tory pathways, including oncogenes and genes involved in
neuropathologies.[40]
At first, we tried to solubilize the guanine following a
modified procedure of Garner, so we performed the di-Boc
protection of G in THF. After a week, we unexpectedly ob-
served that most of the starting base was solubilized and
protected as N1, N2, N2Ј, N9-tetraBoc guanine 15
(Scheme 7). Unfortunately, after several attempts we were
not able to convert 15 into desired bis-Boc-protected base
16; under all conditions a complicated mixture of insepa-
rable compounds was obtained.
Scheme 5. Synthesis of bis-Boc adenine.
Afterward, bis-N-Boc adenine 12 was alkylated with the
use of benzyl bromoacetate in THF by first generating the
anion with sodium hydride (Scheme 6). By using benzyl
bromoacetate instead of methyl bromoacetate it was pos-
sible to recover the corresponding N9-alkylated adenine 13
in higher yield and purity. This procedure yielded only the
expected ethyl adenin-9-ylacetate 13, as was unambiguously
confirmed by NMR spectroscopic analysis.[38] Smooth and
clean cleavage of the benzyl ester by treatment with a cata-
lytic amount of Pd/C quantitatively afforded the desired
bis-N-Boc protected adenylacetic acid 14.
To overcome the low solubility and poor regioselectivity,
we employed 2-amino-6-chloropurine, which is frequently
used as a guanine analogue.[41] Bis-Boc protected guanine
synthon 18 was easily prepared in 75% overall yield by
starting from 2-amino-6-chloropurine after (i) bis-Boc-pro-
tection of the exocyclic amine by using Boc2O/DMAP in
THF and (ii) alkylation of 17 with methyl bromoacetate
(Scheme 8). The synthesis was performed by following the
Scheme 6. Synthesis of bis-Boc-adenylacetic acid.
Put out by the synthesis of compounds 10 and 14, we same methodology as that above for cytosine and ade-
focused our attention on the preparation of bis-Boc-pro- nine.[42]
tected guanine monomer (Figure 2). It is evident from the
It is known that suitably protected 2-amino-6-halopurine
literature that guanine (G) is insoluble in almost all solvents compounds may be converted into protected guanine com-
(water included), and it undergoes direct alkylation to give pounds by several methods involving attack at the 6-halo
a complex reaction mixture including both N9- and N7-al- group by oxygen nucleophiles.[43] Remarkably, the alkoxide
kylated regioisomers.
of 3-hydroxypropionitrile can convert a 6-halo group into a
Scheme 7. Tentative synthesis of bis-Boc-guanine.
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Scheme 8. Synthesis of bis-Boc-6-chloropurine intermediate.
6-carbonyl group and simultaneously hydrolyze the acetate by using NaOH (1 ) in THF at 0 °C for 1 h gave desired
ester group. Two equivalents of alkoxide of 3-hydroxypro- protected guaninylacetic acid 21 in near quantitative yield
pionitrile are consumed in the conversion of the 6-halo (Scheme 9).
group into the 6-carbonyl group, whereas an additional
Unlike the procedure employed by Winssinger, our ap-
equivalent of alkoxide is required to hydrolyze the acetate proach allows bis-Boc-guaninylacetic acid 21 to be synthe-
methyl ester. Unfortunately, by following this procedure we sized by avoiding expensive and toxic reagents such as tri-
are able to recover poorly soluble mono-Boc protected gu- phosgene, which is necessary for the protection of the exo-
aninylacetic acid 19 in only very low yields[23] (Ͻ5%).[44]
cyclic amine group of guanine.[50] Moreover, as is well high-
Alternatively, the displacement of the halogen with ben- lighted by Hudson et al., all bis-N-Boc-protected monomers
zyloxide (with the alcohol as the solvent) from 2-amino-6- (in particular guanine residues) are highly lipophilic, and
chloropurine could be an interesting synthetic route for the this chemical property improves their solubility and reduces
conversion of the 6-chloro group into the 6-oxo function self-aggregation of these residues during oligomerization.
after hydrogenation or acidolysis with TFA. Unfortunately,
In the last step of our strategy, we planned the condens-
this approach was not compatible with our strategy, as it ing reaction (assembling) between N-2-Fmoc-aminoethyl
requires two further steps, exchange of halogen with ben- glycine ester 5 or 6 and the bis-N-Boc protected nucleobase
zyloxide and then benzyl ether removal, and would not acetic acid units 10, 14 and 21 by exploring several coupling
solve the problem of a regioselective subsequent alkylation reagents. The coupling could be accomplished by a number
at N9.[45] Moreover, these strong basic conditions, together of methods. Examples of coupling reagents used to facili-
with the high temperature (65 °C for 16 h) needed to ex- tate this transformation include, but are not limited to, car-
change the 6-halo group for a benzyloxy group, could easily bodiimides such as dicyclohexylcarmodiimide (DCC),
remove the bis-Boc protecting group in the nucleobases.[46] phosphonium salts such as benzotriazol-1-yloxytris(dime-
It is known from the literature that 6-chloropurines react thylamino)phosphonium hexafluorophosphate (BOP) and
with trimethylamine to afford 6-trimethylammonium spe- uronium salts such as HBTU and HATU. Following acti-
cies, which are susceptible to nucleophilic displacement of vation, the side chain moiety is reacted with the amino-
the trimethylammonium group.[47] Therefore, we applied protected backbone to form a PNA synthon. In general,
this method to our substrate. Compound 18 was treated the use of HBTU and HATU, when applied to the synthesis
with 10 equiv. of NMe3 (35% in EtOH[48]) at 0 °C overnight of PNA monomers, enhances the coupling yields and short-
to give an insoluble solid. Strangely and unexpectedly, the ens coupling times.
precipitated solid collected by simple suction filtration was
In place of the above-cited coupling reagents, we pre-
not the expected ammonium salt 19 but desired final gua- ferred to activate the carboxylic acid by use of a mixed an-
nine 20 (Scheme 9).[49] Removal of the methyl ester from 20 hydride. The mixed anhydride has an economical advantage
Scheme 9. Synthesis of bis-Boc-guaninylacetic acid.
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A Practical and Efficient Approach to PNA Monomers
Scheme 10. Synthesis of monomers.
over the more expensive HBTU and HATU reagents, and
A 12mer PNA sequence containing the four natural nu-
it allowed us to prepare the final monomer on a macroscale cleobases was synthesized to check whether the monomers
too.[51]
were compatible with Fmoc-mediated oligomerization and
Examples of sterically hindered acid chlorides and chlo- if the elimination of the protecting groups occurred readily.
roformates used to form “mixed carboxylic anhydrides” The sequence was synthesized by using all three bis-N-Boc
and “mixed carbonic anhydrides”, respectively, include piv- protected monomers 22b, 23b and 24b to give the crude
aloyl chloride (PivCl) and isobutylchloroformate (IBC).[52]
oligomer H-GAGTACGCGCAT-Lys-NH2 under standard
After several coupling tests, our results show that cytosy- conditions.[53] The monomers were linked just as well as the
lacetic acid 10 could couple to amine 5 to afford 22a in conventional Fmoc/Bhoc monomers during the machine-
good yield (88%) by using IBC as the coupling reagent, assisted process. The crude oligomers were analyzed and
whereas the other acids gave poor results under these condi- subsequently purified by RP-HPLC by using a C18 column
tions (Scheme 10). On the contrary, acid 14 and 21 could and a linear gradient of acetonitrile. The identity of the
be effectively coupled to amine 5 to give 23a and 24a (86 oligomers was confirmed by ESI-MS. Analysis of the crude
and 85% yield, respectively) through formation of the piva- product mixture indicated that the desired oligomer repre-
loyl mixed anhydride.
sented Ͼ95% of the material (by HPLC area). Thus, the
In detail, the coupling reagent is added to a cooled THF new monomers were demonstrated to be valid building
solution of bis-Boc protected nucleobases (10, 14 or 21) in blocks for PNA oligomerization.
the presence of a useful nonnucleophilic base such as N-
methylmorpholine (NMM). After stirring at low tempera-
ture (0 °C for PvCl and –10 °C for IBC) for a sufficient time
to permit the formation of mixed anhydride, a THF solu-
Conclusions
tion of Fmoc-protected backbone 5 and NMM was then
In brief, a simple and practical synthesis of orthogonally
added to the cooled solution. The hydrolysis of the resulting protected PNA monomers is described. Protected Aeg
methyl esters 22a–24a was carried out with NaOH (2.5 ) monomers were efficiently prepared by reductive amination
in THF and proceeded smoothly (30 min) to afford the cor- of N-Fmoc-glycinaldehyde with glycine methyl ester and the
responding PNA monomers 22b–24b as white solids in al- subsequent acylation of free amine with N-bis-Boc-pro-
most quantitative yields (Ͼ90%).
tected nucleobases acetic acids. The exocyclic amine group
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5.0 Hz, 2 H) ppm. 13C NMR (CDCl3): δ = 196.4, 156.1, 143.6,
141.2, 127.7, 127.0, 124.9, 119.9, 67.1, 51.5, 47.0 ppm.
of the nucleobases, including guanine, which is notoriously
difficult to handle, was protected with a bis-Boc carbamate
group; this increased the solubility of the nucleobases in the
most common organic solvents. The present protocol allows
all Aeg monomers to be prepared on both the micro- and
macroscale, which avoids or minimizes the use of toxic rea-
gents or solvents, and moreover, cheap starting materials
are used.
Methyl 2-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethylamino)-
acetate (5): A solution of 2 (0.53 g, 1.9 mmol) in MeOH (30 mL)
was added to a solution of glycine methyl ester hydrochloride
(0.24 g, 1.9 mmol) and NEt3 (0.26 mL, 1.9 mmol) in methanol
(50 mL). The mixture was cooled to 0 °C under an atmosphere of
nitrogen for 10 min and 10% Pd/C (0.01 g) was added with vigor-
ous stirring. The reaction mixture was hydrogenated at atmospheric
pressure and room temperature uptake had ceased (overnight). The
catalyst was filtered through a pad of Celite, and the solvent was
evaporated under reduced pressure. The crude product was purified
by flash chromatography (hexane/AcOEt, 1:4) to give backbone
5[14] (0.50 g, 75%) as pale yellow oil. Rf = 0.26 (hexane/AcOEt,
Experimental Section
General Remarks: Starting materials, reagents and dry solvents
were purchased from commercial suppliers and used without fur-
ther purification. Flash-column chromatography was performed on
Merck Kieselgel 60 (230–400 mesh). Thin-layer chromatography
1
1:4). H NMR (CDCl3): δ = 7.71 (d, J = 7.3 Hz, 2 H), 7.57 (d, J
= 7.2 Hz, 2 H), 7.37–7.24 (m, 5 H), 4.39 (d, J = 6.6 Hz, 2 H), 4.17
(t, J = 6.7 Hz, 1 H), 3.65 (s, 3 H), 3.31 (s, 2 H), 3.21 (m, 2 H), 2.66
(t, J = 5.7 Hz, 2 H), 1.90 (s, 1 H) ppm. 13C NMR (CDCl3): δ =
172.6, 156.3, 143.7, 141.0, 127.3, 126.7, 124.7, 119.6, 66.1, 51.5,
49.9, 48.3, 47.0, 40.3 ppm.
1
(TLC) was performed on Merck Kieselgel 60 TLC plates. H and
13C NMR spectra were recorded in the solvents indicated at 300
and 75 MHz, respectively, unless otherwise noted. Chemical shifts
are reported in parts per million (ppm, δ), are measured relative to
tetramethylsilane (0.0 ppm) and referenced to the solvent CDCl3
(7.26 ppm), [D6]DMSO (2.49 ppm) and D2O (4.79 ppm) for 1H
N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-2-methoxy-
2-oxoethanaminium (6): To a precooled solution of 5 (2.83 g,
NMR and CDCl3 (77.0 ppm) and [D6]DMSO (39.5 ppm) for 13C 8.0 mmol) in dry dichloromethane (20 mL) was added HCl (2  in
NMR. Data are reported as follows: chemical shifts, multiplicity (s
= singlet, d= doublet, t = triplet, q = quartet, br. = broad, br. s =
broad singlet, m = multiplet, p = pseudo), coupling constants J
(Hz), relative integration value. Melting points were determined in
open capillary tubes in circulating oil apparatus and are not cor-
rected. High-resolution mass spectra (HRMS) were obtained by
using electron impact (EI) or electrospray (ESI). All the new com-
pounds gave satisfactory elemental analysis.
MeOH, 5 mL, 10 mmol), whereupon the product precipitated out
of the solution immediately. The mixture was allowed to stand at
4 °C overnight in a closed flask. The precipitate was filtered off
with suction and dried for at least 5–6 h under high vacuum to give
6[55] (3.06 g, 98%) as a white solid. M.p. 99–101 °C. Rf = 0.50 (n-
BuOH/AcOH/H2O, 3:1:1). 1H NMR (CD3OD): δ = 7.77 (d, J =
7.5 Hz, 2 H), 7.63 (d, J = 7.5 Hz, 2 H), 7.37 (t, J = 7.5 Hz, 2 H),
7.29 (t, J = 7.5 Hz, 2 H), 4.38 (d, J = 6.3 Hz, 2 H), 4.19 (t, J =
6.3 Hz, 1 H), 4.00 (s, 2 H), 3.81 (s, 3 H), 3.45 (t, J = 5.5 Hz, 2 H),
3.20 (d, J = 5.5 Hz, 2 H) ppm. 13C NMR (CD3OD): δ = 168.0,
159.4, 145.1, 142.6, 128.8, 128.1, 126.1, 120.9, 68.1, 53.5, 49.2, 48.2,
(9H-Fluoren-9-yl)methyl 2-Hydroxyethylcarbamate (1): To a solu-
tion of ethanolamine (1.00 g, 16.37 mmol) in CH2Cl2 (50 mL) was
added saturated Na2CO3 aq. (100 mL) with mechanical stirring.
A solution of 9-fluorenylmethyl chloroformate (Fmoc-Cl, 2.12 g,
8.20 mmol) in CH2Cl2 (15 mL) was added dropwise over 15 min.
The reaction was briskly stirred overnight at room temperature.
The mixture was poured into a separatory funnel, the two layers
were separated and the aqueous layer was extracted again with
CH2Cl2 (3ϫ30 mL). The combined organic layer was washed with
HCl aq. (1 , 3ϫ30 mL) and brine (1ϫ30 mL), dried with anhy-
drous sodium sulfate, filtered and then evaporated to give a white
solid. The resulting crystalline white solid was dried well in vacuo.
48.1, 38.1 ppm. IR (KBr): ν = 3465, 2958, 1749, 1531, 1453, 1250,
˜
742, 588 cm–1.
tert-Butyl 4-[Bis(tert-butoxycarbonyl)amino]-2-oxopyrimidine-
1(2H)-carboxylate (7): To a 100-mL Ar-flushed flask equipped with
a magnetic stir bar and containing cytosine (1.0 g, 9.0 mmol) and
DMAP (0.100 g, 0.9 mmol) was added dropwise dry THF (50 mL).
To the stirred suspension was added Boc2O (7.85 g, 36 mmol) un-
der an Ar atmosphere. The reaction mixture was stirred overnight
at room temperature (TLC analysis indicated the presence of a sin-
gle product and starting solid cytosine was entirely solubilized).
Yield: 2.17 g (93%). Rf = 0.21 (hexane/AcOEt, 1:1). M.p. 143–
1
145 °C. H NMR (CDCl3): δ = 7.76 (d, J = 7.2 Hz, 2 H), 7.58 (d, The excess amount of THF was evaporated, and the crude product
J = 7.2 Hz, 2 H), 7.42–7.25 (m, 5 H), 5.17 (s, 1 H), 4.43 (d, J =
was dissolved in AcOEt (300 mL), washed with HCl aq. (1 ,
6.7 Hz, 2 H), 4.21 (t, J = 7.1 Hz, 1 H), 3.70 (m, 2 H), 3.37–3.31 1ϫ15 mL) and brine (2ϫ15 mL), dried with Na2SO4 and concen-
(m, 2 H) ppm. 13C NMR (CDCl3): δ = 155.3, 144.3, 141.6, 128.0, trated in vacuo to give 7 as a pale yellow oil[56] (3.67 g, 99%). Rf =
127.2, 125.1, 120.1, 68.1, 58.3, 49.0, 43.8 ppm.
0.45 (hexane/AcOEt, 7:3). 1H NMR (CDCl3): δ = 7.97 (d, J =
7.9 Hz, 1 H), 7.07 (d, J = 7.8 Hz, 1 H), 1.60 (s, 9 H), 1.56 (s, 18
H) ppm. 13C NMR (CDCl3): δ = 162.3, 160.0, 150.1, 149.0, 143.3,
96.8, 85.2, 27.5 ppm. HRMS (EI): calcd. for C19H29N3O7 [M]+
411.2006; found 411.1907.
(9H-Fluoren-9-yl)methyl 2-Oxoethylcarbamate (2): To a solution of
alcohol 1 (0.45 g, 1.59 mmol) dissolved in ethyl acetate (25 mL)
was added IBX (1.35 g, 4.82 mmol). The resulting suspension was
immersed in an oil bath set to 80 °C and stirred vigorously open
to the atmosphere. After 4 h (TLC monitoring), the reaction was
cooled to room temperature and filtered through a medium glass
frit. The filter cake was washed with ethyl acetate (3ϫ5 mL), and
the combined filtrates were dried (Na2SO4) and then concentrated
to afford Fmoc-glycinaldehyde 2[54] (0.43 g, 96%, Ͼ95% pure by
1H NMR) as a pale yellow solid. Rf = 0.3 (hexane/AcOEt, 3:7).
Bis-Boc-Cytosine (8): To a solution of 7 (8.08 g, 19.64 mmol) dis-
solved in MeOH (290 mL) was added saturated NaHCO3 aq.
(90 mL). The turbid solution was stirred at 50 °C for 1 h, at which
point clean conversion to bis-Boc protected cytosine was observed
by TLC. After evaporation of MeOH, water (130 mL) was added to
the suspension, and the aqueous layer was extracted with CH2Cl2
(3ϫ100 mL). The organic layer was dried with Na2SO4, filtered
and evaporated to give pure 8 (5.99 g, 99%) as a white solid. Rf =
0.65 (AcOEt). M.p. 320 °C (decomp.). 1H NMR (CDCl3): δ = 7.68
1
M.p. 118°–120 °C. H NMR (CDCl3): δ = 9.61 (s, 1 H), 7.75 (d, J
= 7.5 Hz, 3H, NH), 7.58 (d, J = 7.5 Hz, 2 H), 7.41–7.27 (m, 4 H),
4.41 (d, J = 6.9 Hz, 2 H), 4.21 (t, J = 6.8 Hz, 1 H), 4.11 (d, J =
5792
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5786–5797

A Practical and Efficient Approach to PNA Monomers
(d, J = 7.1 Hz, 1 H), 7.26 (s, 1 H), 7.12 (d, J = 7.1 Hz, 1 H), 1.56
the suspension, and the aqueous layer was extracted with CH2Cl2
(s, 18 H) ppm. 13C NMR (CDCl3): δ = 163.6, 158.4, 149.4, 145.6, (3ϫ100 mL). The organic layer was dried with Na2SO4, filtered
96.7, 84.9, 27.6 ppm. HRMS (ESI): calcd. for C14H22N3O5 [M +
and evaporated to give pure 12 (2.38 g, 96%) a as a white solid. Rf
1
H]+ 312.1554; found 311.1548.
= 0.45 (AcOEt). M.p. 148–149 °C. H NMR (CDCl3): δ = 8.87 (s,
1 H), 8.47 (s, 1 H), 1.50 (s, 18 H) ppm. 13C NMR (CDCl3): δ =
Methyl 2-{4-[Bis(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)-
yl}acetate (9): Sodium hydride (0.33 g, 13.9 mmol) was added to
dry THF (150 mL) in a dry 500-mL round-bottom flask under an
argon atmosphere. The reaction mixture was stirred at 0 °C for a
period of 15–20 min and a solution of bis-Boc cytosine (3.94 g,
12.6 mmol) in dry THF (150 mL) was then added dropwise. To the
rapidly stirred mixture was then adjoined dropwise (over 15–
20 min) methyl bromoacetate (1.32 mL, 13.90 mmol) and DMAP
(0.02 g, 0.16 mmol). After complete addition, the reaction mixture
was stirred overnight whilst warming to room temperature. The
reaction was then quenched by the addition of H2O (0.5 mL,
27.8 mmol). The solvent (THF) was removed, and the residue was
then redissolved in CH2Cl2 (500 mL) and washed with water
(3ϫ100 mL). After evaporation of THF, a light-brown oil was ob-
tained, which was purified by flash chromatography on silica gel
(AcOEt) to give 9 (4.20 g, 86%) as a pale-orange oil. Rf = 0.70
151.9, 151.6, 150.1, 149.1, 144.3, 129.3, 84.5, 27.6 ppm.
Benzyl 2-{6-[Bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetate
(13): To a solution of 12 (1.12 g, 3.34 mmol) in dry THF (60 mL)
cooled in an ice bath for 15 min was added NaH (0.15 g, 5.0 mmol)
under an argon atmosphere whilst stirring. To the rapidly stirred
mixture was then added dropwise (over 10 min) benzyl bromoace-
tate (0.64 mL, 4.01 mmol) and DMAP (0.01 g, 0.08 mmol). After
complete addition, the reaction was stirred overnight whilst warm-
ing to room temperature. The reaction mixture was then quenched
by the addition of H2O (0.18 mL, 10.0 mmol). The solvent (THF)
was removed, and the residue was then redissolved in CH2Cl2
(200 mL) and washed with H2O (3ϫ50 mL). After evaporation of
THF, a light-yellow oil was obtained, which was purified by flash
chromatography on silica gel (hexane/AcOEt, 6:4) to give 13
(1.147 g, 71%) as a pale-yellow oil. Rf = 0.26 (hexane/AcOEt, 6:4).
(AcOEt). 1H NMR (CDCl3): δ = 7.58 (d, J = 7.4 Hz, 1 H), 7.09 1H NMR (CDCl3): δ = 8.83 (s, 1 H), 8.18 (s, 1 H), 7.33 (ps, 5 H),
(d, J = 7.3 Hz, 1 H), 4.59 (s, 2 H), 3.76 (s, 3 H), 1.55 (s, 18 H)
5.20 (s, 2 H), 5.11 (s, 2 H), 1.41 (s, 18 H) ppm. 13C NMR (CDCl3):
ppm. 13C NMR (CDCl3): δ = 198.7, 167.6, 162.7, 149.3, 148.1, δ = 166.4, 153.1, 151.8, 151.3, 149.9, 145.0, 134.2, 128.3, 128.1,
96.4, 84.8, 52.6, 29.5, 27.5 ppm. HRMS (ESI): calcd. for
128.0, 127.8, 83.3, 67.6, 60.0, 44.0, 27.3 ppm. HRMS (ESI): calcd.
C17H26N3O7 [M + H]+ 384.1765; found 384.1751.
for C24H29N5O6 [M]+ 483.2118; found 483.2131.
2-{4-[Bis(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)-
yl}acetic Acid (10): To a solution of 9 (4.7 g, 12.3 mmol) in THF
(40 mL) cooled to 0 °C in an ice bath was added dropwise NaOH
aq. (1 , 13.5 mL, 13.5 mmol). The reaction was then allowed to
stir for about 30 min until TLC indicated complete consumption
of the starting material (the reaction was continuously monitored
by TLC) and then quenched by the addition of H2O (40 mL) and
KHSO4 (portionwise) to pH 2–3. The reaction mixture was poured
into a separatory funnel containing CH2Cl2 (100 mL). The aqueous
layer was further extracted with AcOEt (3ϫ50 mL). The combined
organic fraction was dried with MgSO4, and the solvent was evapo-
rated to dryness in vacuo. The colourless oil was first coevaporated
with CH2Cl2 (3ϫ15 mL) and then concentrated under high vac-
uum for at least 3 h (to an unchanged weight) to give 10 (3.86 g,
85%) as an off-white solid. Rf = 0.1 (hexane/AcOEt, 6:4). M.p.
2-{6-[Bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetic Acid
(14): To a suspension of 13 (0.71 g, 1.47 mmol) in methanol
(40 mL) was added 10% Pd/C (0.05 g) under an argon atmosphere,
and the mixture was hydrogenated until the substrate disappeared
(by TLC, about 8–12 h). The reaction mixture was filtered thought
a pad of Celite, and the solvent was evaporated to give pure 14
(0.57 g, 98%) as a white solid. Rf = 0.1 (hexane/AcOEt, 6:4). M.p.
155–156 °C. 1H NMR (CDCl3): δ = 8.89 (s, 1 H); 8.47 (s, 1 H);
5.10 (s, 2 H); 1.40 (s, 18 H) ppm. 13C NMR (CDCl3): δ = 168.5,
153.1, 152.2, 149.9, 149.8, 146.4, 127.2, 84.2, 44.4, 27.7 ppm.
HRMS (ESI): calcd. for C17H23N5O6 [M]+ 393.1648; found
393.1644.
tert-Butyl 2-[Bis(tert-butoxycarbonyl)amino]-6-chloro-9H-purine-9-
carboxylate (17a): To a 100-mL Ar-flushed flask equipped with a
magnetic stir bar and containing 2-amino-6-chloropurine (1.0 g,
5.9 mmol) and DMAP (0.07 g, 0.59 mmol) was added dropwise dry
THF (50 mL). To the stirred suspension was added Boc2O (5.15 g,
23.6 mmol) under an Ar atmosphere. The reaction mixture was
stirred overnight at room temperature (TLC analysis indicated the
presence of a single product and starting solid 2-mino-6-chloropu-
rine was entirely solubilized). The excess amount of THF was re-
moved, and the crude product was dissolved in AcOEt (300 mL),
washed with HCl aq. (1 , 1ϫ15 mL) and brine (2ϫ15 mL), dried
with Na2SO4 and concentrated in vacuo to give 17a as a white
foam (2.77 g, 99%). Rf = 0.50 (hexane/AcOEt, 6:4). M.p. 51–52 °C.
1H NMR (CDCl3): δ = 8.58 (s, 1 H); 1.69 (s, 9 H); 1.47 (s, 18 H)
ppm. 13C NMR (CDCl3): δ = 153.0, 152.7, 151.2, 149.9, 148.7,
144.3, 139.6, 87.4, 83.2, 27.2 ppm.
1
127°–130 °C. H NMR ([D6]DMSO): δ = 9.82 (s, 1 H), 8.13 (d, J
= 7.2 Hz, 1 H), 6.83 (d, J = 7.2 Hz, 1 H); 4.56 (s, 2 H), 1.49 (s, 18
H) ppm. 13C NMR ([D6]DMSO): δ = 169.1, 161.9, 154.2, 151.2,
149.2, 95.3, 84.4, 54.8, 27.2 ppm. HRMS (EI): calcd. for
C16H24N3O7 [M + H]+ 370.1614; found 370.1610.
tert-Butyl 6-[Bis(tert-butoxycarbonyl)amino]-9H-purine-9-carboxyl-
ate (11): To a 100-mL Ar-flushed flask equipped with a magnetic
stir bar and containing adenine (1.00 g, 7.4 mmol) and DMAP
(0.08 g, 0.74 mmol) was added dry THF (50 mL). To the stirred
suspension was added Boc2O (6.46 g, 29.6 mmol) under an Ar at-
mosphere. The reaction mixture was stirred overnight at room tem-
perature, at which point TLC analysis indicated the presence of a
single product. The excess amount of THF was removed to give 11
(3.22 g, 99%) as a yellow oil. Rf = 0.45 (hexane/AcOEt, 7:3). M.p.
Bis-Boc-2-amino-6-chloropurine (17b): To a solution of 17a (2.8 g,
6.0 mmol) in MeOH (100 mL) was added saturated NaHCO3 aq.
(50 mL). The turbid solution was stirred at 50 °C for 1 h, at which
point clean conversion to bis-Boc protected adenine was observed
by TLC. After evaporation of MeOH, H2O (40 mL) was added to
1
54–55 °C. H NMR (CDCl3): δ = 9.02 (s, 1 H), 8.53 (s, 1 H), 1.72
(s, 9 H), 1.44 (s, 18 H) ppm. 13C NMR (CDCl3): δ = 156.4, 153.6,
152.1, 150.8, 149.6, 142.9, 129.2, 87.1, 83.5, 27.3 ppm.
Bis-Boc-Adenine (12): To a solution of 11 (3.22 g, 7.4 mmol) dis-
solved in MeOH (80 mL) was added saturated NaHCO3 aq. the suspension, and the aqueous layer was extracted with CH2Cl2
(40 mL). The turbid solution was stirred at 50 °C for 1 h, at which
point clean conversion to bis-Boc protected adenine was observed
by TLC. After evaporation of MeOH, water (50 mL) was added to
(3ϫ80 mL). The organic layer was dried with Na2SO4, filtered and
evaporated to give pure 17b (2.20 g, 99%) as a waxy solid. Rf =
0.60 (AcOEt). H NMR (CDCl3): δ = 8.41 (s, 1 H); 1.49 (s, 18 H)
1
Eur. J. Org. Chem. 2008, 5786–5797
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5793

A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta, G. Nieddu
ppm. 13C NMR (CDCl3): δ = 155.7, 150.6, 150.3, 148.0, 147.2, temperature remained –15 °C. Stirring was continued for an ad-
FULL PAPER
127.5, 83.5, 27.3 ppm.
ditional 15 min. A reaction temperature between –10 and –15 °C is
recommended, where the reaction occurs immediately upon ad-
dition of NMM, yet prevents the mixed anhydride from decompos-
ing too rapidly. Compound 5 (0.83 g, 2.35 mmol), meanwhile, was
dissolved in dry THF (15 mL) and cooled to 0 °C. This latter solu-
tion was upon completion of the activation added at a rate that
maintained the temperature around –15 °C (about 10 min). Stirring
was subsequently continued for 30 min at –10 °C. The reaction
mixture was warmed to room temperature and further stirred at
this temperature overnight. After removal of the solvent, the or-
ganic residue was redissolved in AcOEt (250 mL) and then washed
with HCl aq. (1 , 3 ϫ 50 mL) and saturated NaHCO3 aq.
(3ϫ50 mL). The organic phase was then dried with Na2SO4, and
the solvent was evaporated under vacuum. The crude reaction
product was purified by flash chromatography (AcOEt). The com-
bined fractions were dried to give 22a as a pale-yellow oil (1.32 g,
Methyl 2-{2-[Bis(tert-butoxycarbonyl)amino]-6-chloro-9H-purin-9-
yl}acetate (18): Sodium hydride (0.2 g, 4.9 mmol) was added to dry
THF (100 mL) in a dry 500-mL round-bottom flask under an ar-
gon atmosphere. The reaction mixture was stirred at 0 °C for a
period of 10–15 min, and a solution of bis-Boc 2-amino-6-chlorpu-
rine (1.64 g, 4.43 mmol) in dry THF (100 mL) was added dropwise.
To the rapidly stirring mixture was then adjoined dropwise (over 10
to 15 min) methyl bromoacetate (0.46 mL, 4.9 mmol) and DMAP
(0.01 g, 0.08 mmol). After complete addition, the reaction was
stirred overnight whilst warming to room temperature. The reac-
tion mixture was then quenched by the addition of H2O (0.18 mL,
9.8 mmol). The solvent (THF) was removed, and the residue was
then redissolved in CH2Cl2 (300 mL) and washed with water
(3ϫ100 mL). After evaporation of THF, a light-brown oil was ob-
tained, which was purified by flash chromatography on silica gel
(AcOEt/hexane, 7:3) to give 18 (1.53 g, 78%) as a pale-yellow oil.
1
88%). H NMR (CDCl3): δ = 7.73 (d, J = 7.5 Hz, 2 H), 7.60 (t, J
= 7.4 Hz, 2 H), 7.44–7.27 (m, 5 H), 7.06 (t, J = 7.3 Hz, 1 H), 6.16
(t, J = 7.4 Hz, 1 H), 4.58 (s, 2 H), 4.40 (d, J = 7.2 Hz, 2 H), 4.19
(t, J = 7.5 Hz, 1 H), 4.03 (s, 2 H), 3.71 (s, 3 H), 3.59–3.34 (m, 4
H), 1.53 (s, 18 H) ppm. 13C NMR (CDCl3): δ = 170.3, 167.5, 162.9,
156.9, 155.3, 155.2, 149.6, 149.4, 144.1, 141.4, 127.9, 127.3, 126.9,
125.3, 120.1, 96.3, 85.1, 66.9, 53.0, 52.6, 49.7, 49.1, 47.3, 39.5,
27.8 ppm. HRMS (ESI): calcd. for C36H44N5O10 [M]+ 706.3083;
found 706.3095.
1
Rf = 0.44 (AcOEt/hexane, 7:3). H NMR (CDCl3): δ = 8.31 (s, 1
H), 5.10 (s, 2 H), 3.82 (s, 3 H), 1.43 (s, 18 H) ppm. 13C NMR
(CDCl3): δ = 166.7, 152.6, 151.8, 150.9, 150.2, 146.6, 129.3, 83.5,
52.9, 44.3, 27.6 ppm. HRMS (ESI): calcd. for C18H24ClN5O6 [M]+
441.1415; found 441.1418.
Methyl 2-{2-[Bis(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-
9(6H)-yl}acetate (20): To a stirred ethanolic solution of NMe3
(35% in EtOH/H2O, 95:5; 12 mL, 2 mmol) was portionwise added
18 (1.07 g, 2.42 mmol), and the reaction mixture was allowed to
stir at room temperature overnight.[57] The mixture was centri-
fuged, and the crude precipitate was filtered off and washed with
anhydrous Et2O to give 20 as a white solid, which was further dried
under high vacuum (2 mbar) for at least 3 h (0.69 g, 67%). M.p.
2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-2-{4-[bis-
(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)-yl}acetamido]-
acetic Acid (22b): Methyl ester 22a (0.71 g, 1 mmol) dissolved in
THF (2 mL) and cooled to 0 °C was subjected to hydrolysis with
NaOH aq. (2.5 , 4 mL, 10 equiv.). The mixture was stirred at 0 °C
for about 30 min, at which point the TLC showed complete disap-
pearance of the starting ester. Cold H2O (10 mL) was added to the
reaction mixture, which was first extracted with Et2O (3ϫ5 mL)
and then acidified to pH 3 with KHSO4 (at 0 °C, the flask was
placed in an ice bath). The aqueous solution was extracted with
AcOEt (3ϫ25 mL), and the combined organic layer was washed
with brine (2 ϫ 25 mL). The organic solution was dried with
MgSO4 overnight, and the solvent was then evaporated to dryness
in vacuo to give the desired final monomer 22b (0.622 g, 90%) as
1
180–182 °C (decomp.). H NMR (CDCl3): δ = 7.81 (s, 1 H), 4.95
(s, 2 H), 3.76 (s, 3 H), 1.45 (s, 18 H) ppm. 13C NMR (CDCl3): δ
= 167.6, 155.1, 152.4, 151.6, 151.2, 139.0, 117.8, 82.3, 52.5, 43.7,
27.7 ppm. HRMS (ESI): calcd. for C18H26N5O7 [M + H]+
424.1827; found 424.1830.
2-{2-[Bis(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-9(6H)-
yl}acetic Acid (21): To a solution of 20 (1.69 g, 4.0 mmol) in THF
(25 mL) cooled to 0 °C in an ice bath was added dropwise NaOH
aq. (1 , 4.4 mL, 4.4 mmol). The reaction mixture was then allowed
to stir for about 30 min until TLC indicated complete consumption
of the starting material (the reaction was continuously monitored
by TLC) and then quenched by the addition of H2O (22 mL) and
KHSO4 (portionwise) to pH 3. The reaction mixture was poured
into a separatory funnel containing CH2Cl2 (100 mL). The aqueous
layer was further extracted with AcOEt (3ϫ50 mL). The combined
organic fraction was dried with MgSO4, and the solvent was evapo-
rated to dryness in vacuo. The colourless oil was first coevaporated
with CH2Cl2 (3ϫ15 mL) and then concentrated under high vac-
uum for at least 3 h (to an unchanged weight) to give 21 (1.60 g,
1
an off-white solid. M.p. 110–112 °C. H NMR (CDCl3): δ = 7.73
(d, J = 7.7 Hz, 2 H), 7.60–7.48 (m, 3 H), 7.37–7.23 (m, 5 H), 7.14–
7.07 (m, 1 H), 6.40 (t, J = 7.3 Hz, 1 H), 4.67 (s, 2 H), 4.33 (d, J =
7.4 Hz, 2 H), 4.21–4.17 (m, 1 H), 4.00 (s, 2 H), 3.52–3.38 (m, 4 H),
1.51 (s, 18 H) ppm. 13C NMR (CDCl3): δ = 171.1, 166.9, 162.8,
156.9, 156.3, 149.6, 149.2, 143.8, 141.1, 127.5, 127.0, 126.3, 125.1,
119.8, 97.0, 85.3, 66.9, 53.3, 52.9, 48.5, 47.0, 38.9, 27.5 ppm.
HRMS (ESI): calcd. for C35H41N5O10Na [M]+ 714.2751; found
714.2756.
Methyl 2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-
2-{6-[bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetamido]-
acetate (23a): To a solution of 14 (0.98 g, 2.5 mmol) in dry acetoni-
trile (80 mL) was added in one portion freshly distilled N-methyl-
morpholine (NMM, 0.52 mL, 5 mmol) at room temperature, and
the resulting mixture was stirred for 10 min. The mixture was co-
oled to 0 °C, and pivaloyl chloride (PivCl, 0.33 mL, 2.5 mmol) was
added dropwise under an argon atmosphere. The reaction mixture
was further stirred for 30 min at 0 °C. Then, 5 (1 g, 2.8 mmol) was
added under vigorous stirring to the starting reaction mixture, and
1
98%) as an off-white solid. H NMR (CDCl3): δ = 7.93 (s, 1 H),
4.91 (s, 2 H), 1.43 (s, 18 H) ppm. 13C NMR (CDCl3): δ = 168.9,
156.9, 155.1, 151.2, 151.4, 140.1, 117.4, 82.8, 44.2, 27.8 ppm.
HRMS (ESI): calcd. for C17H24N5O7 [M + H]+ 410.1670; found
410.1668.
Methyl 2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-
2-{4-[bis(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)-
yl}acetamido]acetate (22a): Under an atmosphere of argon at
–15 °C, isobutylchloroformate (IBC, 0.28 mL, 2.14 mmol) was the resulting mixture was stirred overnight at room temperature.
dosed to a solution of 10 (0.79 g, 2.14 mmol) in dry THF (60 mL).
Subsequently, N-methyl morpholine (NMM, 0.44 mL, 4.28 mmol)
in dry THF (3 mL) was added dropwise at such a rate that the
The solution was diluted with ethyl acetate (300 mL) and then
washed with brine (2ϫ50 mL). The aqueous layer was back ex-
tracted with ethyl acetate (4ϫ50 mL). The combined organic layer
5794
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5786–5797

A Practical and Efficient Approach to PNA Monomers
was washed with HCl aq. (1 , 3ϫ50 mL), brine (1ϫ50 mL), satu-
rated NaHCO3 aq. (3ϫ50 mL) and brine (1ϫ50 mL) and then
dried with MgSO4, and the solvent was evaporated to dryness in
vacuo. The crude material was purified by flash chromatography
(AcOEt) to give desired monomer 23a (1.57 g, 86%) as a pale-yel-
HRMS (ESI): calcd. for C37H44N7O10 [M + H]+ 746.3144; found
746.3130.
2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-2-{2-[bis-
(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-9(6H)-yl}acetamido]-
acetic Acid (24b): Methyl ester 24a (0.75 g, 1 mmol) dissolved in
THF (2 mL) and cooled to 0 °C was subjected to hydrolysis with
NaOH aq. (2.5 , 4 mL, 10 equiv.). The mixture was stirred at 0 °C
for about 30 min, at which point the TLC showed complete disap-
pearance of the starting ester. Cold H2O (10 mL) was added to the
reaction mixture, which was first extracted with Et2O (3ϫ5 mL)
and then acidified to pH 3 with KHSO4 (at 0 °C, the flask was
placed in an ice bath). The aqueous solution was extracted with
AcOEt (3ϫ25 mL), and the combined organic layer was further
washed with brine (2ϫ25 mL). The organic solution was dried
with MgSO4 overnight, and the solvent was then evaporated to
dryness in vacuo to give the desired final monomer 24b (0.680 g,
93%) as an off-white solid. M.p. 255 °C (decomp.). 1H NMR
(CDCl3): δ = 7.99 (s, 1 H), 7.69 (t, J = 7.4 Hz, 3 H), 7.53 (t, J =
7.1 Hz, 2 H), 7.36–7.22 (m, 5 H), 5.06 (s, 2 H), 4.30 (t, J = 6.8 Hz,
2 H), 4.17–4.12 (m, 1 H), 4.05 (s, 2 H), 3.55–3.34 (m, 4 H), 1.42 (s,
18 H) ppm. 13C NMR (CDCl3): δ = 173.1, 167.5, 156.5, 155.1,
152.1, 151.4, 143.8, 143.6, 141.0, 140.2, 127.5, 126.8, 125.0, 124.7,
119.7, 117.7, 82.4, 66.6, 53.3, 49.3, 46.2, 38.2, 29.4, 27.7 ppm.
HRMS (ESI): calcd. for C36H41N7O10 [M + H]+ 731.2915; found
731.2920.
1
low solid. Rf = 0.16 (AcOEt). M.p. 84°–85 °C. H NMR (CDCl3):
δ = 8.17 (s, 1 H), 8.05 (s, 1 H), 7.76–7.69 (m, 3 H), 7.60–7.56 (m,
2 H), 7.38–7.26 (m, 4 H), 5.06 (s, 2 H), 4.52 (d, J = 6.7 Hz, 2 H),
4.21 (t, J = 6.1 Hz, 1 H), 4.04 (s, 2 H), 3.72 (s, 3 H), 3.63–3.58 (m,
2 H), 4.45–3.41 (m, 2 H), 1.41 (s, 18 H) ppm. 13C NMR (CDCl3):
δ = 170.2, 169.2, 156.5, 153.3, 151.8, 150.4, 150.0, 143.7, 143.5,
141.2, 127.6, 126.9, 125.0, 119.9, 87.9, 67.1, 50.1, 49.2, 47.1, 39.0,
38.8, 28.2 ppm. HRMS (ESI): calcd. for C37H44N7O9 [M + H]+
730.7862; found 730.7853.
2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-2-{6-[bis-
(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetamido]acetic Acid
(23b): Methyl ester 23a (0.729 g, 1 mmol) dissolved in THF (2 mL)
cooled to 0 °C was subjected to hydrolysis with NaOH aq. (2.5 ,
4 mL, 10 equiv.). The mixture was stirred at 0 °C for about 30 min,
at which point the TLC showed complete disappearance of the
starting ester. Cold H2O (10 mL) was added to the reaction mix-
ture, which was first extracted with Et2O (3ϫ5 mL) and then acidi-
fied to pH 3 with KHSO4 (at 0 °C, the flask was placed in an ice
bath). The aqueous solution was extracted with AcOEt
(3ϫ25 mL), and the combined organic layer was further washed
with brine (2 ϫ 25 mL). The organic solution was dried with
MgSO4 overnight, and the solvent was evaporated to dryness in
vacuo to give the desired final monomer 23b (0.672 g, 94%) as an
off-white solid. M.p. 103–104 °C. 1H NMR (CDCl3): δ = 8.27 (s, 1
H), 8.16 (s, 1 H), 7.74–7.65 (m, 3 H), 7.58–7.48 (m, 2 H), 7.36–7.23
(m, 4 H), 5.14 (s, 2 H), 4.43 (d, J = 7.4 Hz, 2 H), 4.20–4.17 (m, 1
H), 4.01 (s, 2 H), 3.51–3.35 (m, 4 H), 1.38 (s, 18 H) ppm. 13C NMR
(CDCl3): δ = 176.4, 171.4, 157.2, 153.5, 152.1, 150.1, 149.8, 143.7,
143.6, 141.1, 127.6, 127.0, 125.4, 125.0, 119.8, 83.9, 66.8, 48.8, 48.3,
47.0, 39.5, 38.7, 27.6 ppm. HRMS (ESI): calcd. for C36H41N7O9Na
[M + Na]+ 738.2863; found 738.2850.
Supporting Information (see footnote on the first page of this arti-
cle): 1H and 13C NMR spectra for compounds 1–24b; HPLC spec-
trum for the PNA sequence.
Acknowledgments
This work was financially supported by the University of Sassari,
Fondazione Banco di Sardegna and MIUR (Rome) within the pro-
ject PRIN 2005: “Structure and Activity Studies of DNA Quadru-
plexes through the Exploitation of Synthetic Oligonucleotides
and Analogues”. For a closer examination, see: http://www.
ricercaitaliana.it/prin/unita_op_en-2005030447_004.htm.
Methyl 2-[N-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl)-
2-{2-[bis(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-9(6H)-yl}-
acetamido]acetate (24a): To a solution of 21 (1.29 g, 3.15 mmol) in
dry acetonitrile (100 mL) was added in one portion N-M ethyl-
morpholine (NMM, 0.65 mL, 6.3 mmol) at room temperature, and
the resulting mixture was stirred for 10 min. The mixture was co-
oled to 0 °C, and pivaloyl chloride (PivCl, 0.42 mL, 3.15 mmol)
was added dropwise under an argon atmosphere. The reaction mix-
ture was further stirred for 30 min at 0 °C. Then, 5 (1.23 g,
3.45 mmol) was added under vigorous stirring to the starting reac-
tion mixture, and the resulting mixture was stirred overnight at
room temperature. The solution was diluted with ethyl acetate
(350 mL) and then washed with brine (2ϫ50 mL). The aqueous
layer was back extracted with AcOEt (4ϫ50 mL). The combined
organic layer was washed with HCl aq. (1 , 3 ϫ50 mL), brine
(1 ϫ 50 mL), saturated NaHCO3 aq. (3 ϫ 50 mL) and brine
(1ϫ50 mL) and then dried with MgSO4, and the solvent was evap-
orated to dryness in vacuo. The crude material was purified by
flash chromatography (AcOEt) to give the desired monomer 24a
[1] P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science
1991, 254, 1497.
[2] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M.
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[6] For more about the application, properties and standard syn-
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Chem. Res. 1999, 32, 624 and the references cited therein.
[7] a) A. Porcheddu, G. Giacomelli, Curr. Med. Chem. 2005, 12,
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hedron 1995, 51, 6179; f) D. W. Will, G. Breipohl, D. Langner,
1
(2.0 g, 85%) as a pale-yellow oil. H NMR (CDCl3): δ = 7.87 (s, 1
H), 7.73 (t, J = 7.1 Hz, 3 H), 7.55 (t, J = 6.9 Hz, 2 H), 7.38–7.24
(m, 4 H), 4.96 (s, 2 H), 4.32 (d, J = 6.9 Hz, 2 H), 4.17–4.11 (m, 1
H), 4.05 (s, 2 H), 3.72 (s, 3 H), 3.61–3.29 (m, 4 H), 1.46 (s, 18 H)
ppm. 13C NMR (CDCl3): δ = 169.7, 167.1, 157.7, 156.6, 155.1,
152.2, 151.5, 143.6, 141.1, 139.5, 127.5, 126.9, 125.8, 124.9, 119.8,
117.7, 82.7, 66.6, 52.3, 49.2, 48.6, 47.0, 38.8, 29.5, 27.8 ppm.
Eur. J. Org. Chem. 2008, 5786–5797
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5795

A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta, G. Nieddu
FULL PAPER
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days and used as needed, although loss of the Fmoc group
does occur with time.[10]
T. Stafforst, U. Diederichsen, Eur. J. Org. Chem. 2007, 681.
Working on macroscale (5–10 g), a more attractive solution is
to simply wash the crude reaction mixture with dilute aqueous
hydrochloric acid and store at –20 °C overnight. This results
in precipitation of the pure hydrochloride salt of 6 as a white
powder.
DMF has been linked to cancer in humans, and it is thought
to cause birth defects. In some sectors of industry women are
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Y. Wu, J.-C. Xu, Tetrahedron 2001, 57, 8107.
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Wiethe, S. A. Noble, Tetrahedron 1995, 51, 6179.
Fmoc/Bhoc PNA monomers are commercially available from
ASM Research Chemicals: www.asm-research-chemicals.com/
index.
The modular nature of PNA is particularly amenable to struc-
tural variation, and this has led to the synthesis of a wide vari-
ety of modified PNAs.
For a more detailed comparison between N-Bhoc and N-bis-
Boc nucleobase protective groups see Hudson’s paper, page
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G. Breiphol, J. Knolle, D. Langner, G. O’Malley, E. Uhlmann,
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The no-observed-adverse-effect level of tetrahydrofuran admin-
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and reproductive performance, and 3000 ppm for systemic par-
ental and developmental toxicity. J. Hellwiga, C. Gembardta,
S. Jastib, Food Chem. Toxicol. 2002, 40, 1515.
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This latter method is better because of its simplicity.
a) J. Kjellberg, M. Liljenberg, N. G. Johansson, Tetrahedron
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[42]
[43]
The alkylation reaction was found to be regioselective for N-9,
as no N-7 isomer was detected by 1H and 13C NMR spec-
troscopy.
a) M. Ashwell, C. Bleasdale, B. T. Golding, I. K. O’Neill, J.
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This procedure was conducted in duplicate and did not always
give reproducible results.
From our experience, most displacement reactions with alk-
oxide from 2-amino-6-chloropurine are rather elaborate and
give the desired guanine in low yields. Hudson and Wojcie-
Winssinger et. al. investigated six types of protecting groups
for the terminal nitrogen atom of the Aeg residue (Alloc, Teoc,
4-N3Cbz, Fmoc, 4-OTBSCbz and Azoc) and five protecting
groups on the nucleobases (Cl-Bhoc, FBhoc, Teoc, 4-OMeCbz
and Boc). The authors observed that the most suitable combi-
nation of protecting groups was mono-Boc-protected nucleo-
bases with either Azoc or Fmoc protecting groups for the ter-
minal nitrogen atom.
[44]
[45]
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Eur. J. Org. Chem. 2008, 5786–5797

A Practical and Efficient Approach to PNA Monomers
chowski (a team with a deep experience in PNA synthesis) in
their seminal paper suggest the procedure outline by Kunz (C.
Barth, O. Seitz, H. Z. Kunz, Naturforsch. 2004, 59b, 802) to
climb this obstacle. Honestly, we have no direct experience with
this last procedure.
[51]
[52]
This procedure was successfully applied for final quantities of
monomers up to 5 g.
These mixed anhydrides overcome some waste problems, as the
only side products are, respectively, pivalic acids (it could be
recovered and recycled) or CO2 and isobutyl alcohol (two vola-
tile compounds). Moreover, IBC and pivaloyl chloride are
cheap and couple easily to the acid. These last points of view
are not marginal if our goal is the preparation of all monomers
on macroscale.
Used protocols were based on those provided in: R. Casale,
I. S. Jensen, M. Egholm, “Synthesis of PNA Oligomers by
Fmoc Chemistry” in Peptide Nucleic Acids: Protocols and Ap-
plications (Eds.: P. E. Nielsen), 2nd ed., Garland Science, 2003.
[46] After several attempts and two years of unsuccessful research
we still were at a dead end.
[47] Trimethylammonium is a better leaving group than chloride in
the addition–elimination mechanism leading to displacement
of the 6-substituent by a nucleophile.
[48] The ethanolic solution of NMe3 is not dry and contains 5%
H2O (EtOH/H2O, 95:5).
[49] The structure of the collected solid was confirmed by detailed
spectroscopic, mass and elemental analysis. The same reaction
was conducted in duplicate and with increasing quantities of
the substrate (up to 2 g); we never observed the presence of a
6-dimethylamino byproduct. For a closer examination, see the
NMR spectroscopic analysis in the Supporting Information.
[50] In the protocol outlined by Winssinger to prepare N-Boc-pro-
tected guanine, the exocyclic nitrogen atom was converted into
an isocyanate, which was then treated with tBuOH. Unlike
other nucleobases, the lower reactivity of 6-amino-2-chloropu-
rine necessitated the use of triphosgene for isocyanate forma-
tion.
[53]
[54]
J. D. More, N. S. Finney, Org. Lett. 2002, 4, 3001.
[55] For the preparation and full characterization of HCl·Fmoc-
Aeg-OMe, see the very interesting paper of Stafforst and Died-
erichsen in ref.[33]
[56] The residue was put under high vacuum for at least 2–3 h to
remove all traces of tBuOH.
[57] An off-white solid started to precipitate just after 30 min.
Received: September 11, 2008
Published Online: October 29, 2008
Eur. J. Org. Chem. 2008, 5786–5797
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5797