Expanding the scope and orthogonality of PNA synthesis

Article abstract of DOI:10.1002/ejoc.200800141

Peptide nucleic acids (PNAs) hybridize to natural oligonucleotides according to Watson and Crick base-pairing rules. The robustness of PNA oligomers and ease of synthesis have made them an attractive platform to encode small or macromolecules for microarr

Full text of DOI:10.1002/ejoc.200800141

FULL PAPER
DOI: 10.1002/ejoc.200800141
Expanding the Scope and Orthogonality of PNA Synthesis
Srinivasu Pothukanuri,[a] Zbigniew Pianowski,[a] and Nicolas Winssinger*[a]
Keywords: Peptides / Nucleic acids / Combinatorial chemistry / Protecting groups
Peptide nucleic acids (PNAs) hybridize to natural oligonucle-
otides according to Watson and Crick base-pairing rules. The
robustness of PNA oligomers and ease of synthesis have
made them an attractive platform to encode small or macro-
molecules for microarraying purposes and other applications
based on programmable self assembly. A cornerstone of
these endeavors is the orthogonality of PNA synthesis with
other chemistries. Herein, we present a thorough investiga-
tion of six types of protecting groups for the terminal nitrogen
atom (Alloc, Teoc, 4-N3Cbz, Fmoc, 4-OTBSCbz, and Azoc)
and five protecting groups on the nucleobases (Cl-Bhoc, F-
Bhoc, Teoc, 4-OMeCbz, and Boc).
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
to extend the orthogonality of PNA chemistry by extending
the arsenal of protecting groups (1, Figure 1) and report
practical procedures for the large-scale preparation of PNA
monomers 1 from protected N-(2-aminoethyl)glycine back-
bone 2 and acid 3.
Introduction
Peptide nucleic acids[1,2] (PNAs) are unnatural oligonu-
cleotide analogues in which the entire sugar–phosphate
backbone is replaced by an achiral and uncharged pseudo-
peptide backbone consisting of N-(2-aminoethyl)glycine
units to which the nucleobases are attached through methyl-
ene carbonyl linkers. PNAs hybridize preferably in the anti-
parallel alignment to complementary DNA/RNA/PNA in a
sequence-specific manner and such a duplex exhibits higher
thermal stability than their natural counterparts DNA/
DNA and DNA/RNA.[3,4] Additionally, PNAs are remark-
ably resistant to enzymatic degradation, which makes them
an ideal tool for the development of therapeutics, reagents
in molecular biology, and diagnostics. From a chemistry
standpoint, PNAs do not suffer from facile depurination as
ribose-based oligonucleotides do and their oligomerization
is based on versatile peptide bond formation, which thus
facilitates the parallel synthesis of PNA appended to func-
tional molecules.[5] We have been particularly interested in
the use of PNA-encoded small-molecule libraries that can
be converted into a microarray format by hybridization to
DNA arrays.[6] PNA monomers for both Boc- or Fmoc-
based synthesis are commercially available with Cbz or
Bhoc-protected nucleobases, respectively. Alternative pro-
tecting groups for the terminal nitrogen atom include
Mmt,[7] Dts,[8] Dmt,[9] N3,[10] Alloc,[11] Dde,[12] Nvoc,[13] and
Bts.[14] Beyond the commercially available monomers, the
most frequently used protecting groups for nucleobases are
Cbz, Mmt, and acyl groups.[15] Herein, we report our efforts
Figure 1. General structure of PNA monomer 1 and its synthetic
precursors 2 and 3. PG = protecting group; B = nucleobases; R =
H, Me, or Bn.
Results and Discussion
The nucleobases protected with Bhoc, Cl-Bhoc, F-Bhoc,
4-OMeCbz, Teoc, or Boc groups were obtained in three-to-
four steps as shown in Scheme 1. The cytosine (4), adenine
(8), and 6-amino-2-chloropurine (11) heterocycles were al-
kylated with benzyl 2-bromoacetate and the exocyclic nitro-
gen atoms were converted into an isocyanate, which was
then engaged in a reaction with different alcohols 5 to ob-
tain carbamate-protected compounds 6, 9, and 12. The
lower reactivity of 6-amino-2-chloropurine necessitated tri-
phosgene for isocyanate formation, whereas the reaction
[a] Organic and Bioorganic Chemistry Laboratory, Institut de Sci-
ence et Ingénierie Supramoléculaires, Université Louis Pasteur,
8 allée Gaspard Monge 67000 Strasbourg, France
Fax: +33-3-90-24-51-12
E-mail: winssinger@isis.u-strasbg.fr
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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S. Pothukanuri, Z. Pianowski, N. Winssinger
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Scheme 1. Synthesis of protected nucleobases 7, 10, and 13. Reagents and conditions: (a) tBuOK (1.15 equiv.), DMF, 100 °C for 2 h, then
benzyl 2-bromoacetate (1.12 equiv.), 0 °C to 23 °C, 12 h, 92%; (b) CDI (1.6 equiv.), ROH 5 (1.3–5.0 equiv.), DMF, 60–80 °C to 23 °C,
12 h, 72–94%; (c) LiOH (4–10 equiv.), 0 °C or 23 °C, 10 min, 69–94%; (d) NaH (1.2 equiv.), benzyl 2-bromoacetate (1.1 equiv.), DMF
0 °C to 23 °C, 12 h, 84%; (e) CDI (1.5–3 equiv.), ROH (1.5–3 equiv.), DMF, 105 °CǞ23 °C, 12 h, 51%; (f) LiOH (18 equiv.), MeCN/
H2O/EtOH, 10 °C, 10 min, 73% (R-benzylic); or H2, Pd/C, EtOH, 23 °C, 91% (R = tBu); (g) K2CO3 (1.5 equiv.), DMF, 85 °C for 30 min,
then benzyl 2-bromoacetate (1.1 equiv.), 0 °C to 23 °C, 12 h, 89%; (h) triphosgene (0.36 equiv.), EtiPr2N (2.2 equiv.), ROH (1.2–3.0 equiv.),
THF, 0 °C to 23 °C, 12 h, 87%; (i) NaH (5.0 equiv.), 3-hydroxypropionitrile (5.0 equiv.), THF, –78 °CǞ23 °C, 12 h, 76–86%.
proceeded smoothly with carbonyldiimidazole for the ade-
Suitably protected backbones 17 were acylated with nu-
nine and cytosine heterocycles. The benzyl esters were then cleobase acetic acids 3 (7, 10, and 13 and thymine acetic
hydrolyzed to obtain suitably derivatized nucleobases 7, 10, acid) as shown in Scheme 3. In general, the acylation with
and 13. Although the benzyl ester of adenine 9f could be the pyrimidines (thymine acetic acid and cytosine acetic
conveniently cleaved under reductive conditions (H2, Pd/C), acid 7) could be carried out by using a variety of activation
hydrogenolysis of cytosine 6f led to partial reduction of the methods (HBTU, DIC/HOBt, TSTU, or Piv-Cl), whereas
heterocycle. The hydrolysis of compounds 12 required the the activation of the purines (10 and 13) could not be
concomitant conversion of the 6-chloro functionality into achieved with carbodiimide reagents. Nevertheless, acti-
the ketone, which was most conveniently achieved with an vation in the form of N-benzotriazole esters (HBTU), N-
excess amount of the alkoxide of 3-hydroxypropionitrile, succinimate esters (TSTU), or mixed anhydrides (pivaloyl
which undergoes substitution followed by β-elimination.
chloride) were effective. Hydrolysis of the methyl or benzyl
Suitably protected N-(2-aminoethyl)glycine backbones ester afforded acid 21 in good yield except for the com-
17a–e were prepared as shown in Scheme 2. Phenyl carbon- pound bearing the 4-OTBSCbz group (17c), which was par-
ates 15, obtained from the reaction of phenyl chloroformate tially hydrolyzed during the reaction. Interestingly, coupling
and alcohols 14, were used for the monoprotection of ethyl- of activated heterocyclic acetic acids 3 with 17a–e pro-
endiamine[16] followed by alkylation of the remaining pri- ceeded smoothly with the glycine moiety of 17 protected as
mary amine with methyl or benzyl 2-bromoacetate thus af- a methyl or benzyl ester (R2), whereas the equivalent reac-
fording 17a–d. Azoc[17]-protected backbone 17e was pre- tion with 17f in the form of a benzyl ester (not shown) gave
pared by reductive amination of glycine benzyl ester 20 and poor results. We found it preferable to carry out the acyl-
aldehyde 19, which was in turn obtained in three steps from ation with unprotected 17f by using a mild activation
ethanolamine.
method such as TSTU, as the use of stronger activation
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Expanding the Scope and Orthogonality of PNA Synthesis
Scheme 2. Synthesis of protected N-(2-aminoethyl)glycine 17. Reagents and conditions: (a) phenyl chloroformate (1.0 equiv.), pyridine
(1.25 equiv.), CH2Cl2, 23 °C, 4 h, quantitative; (b) 16 (1.0 equiv.), EtOH, 23 °C, 12 h, 54–68%; (c) benzyl 2-bromoacetate (1.0 equiv.) or
methyl 2-bromoacetate (1.0 equiv.), Et3N (1.0 equiv.), CH2Cl2, 0 °C, 4 h, 40–63%; (d) ethanolamine (2.0 equiv.), CH2Cl2, 18 (1.0 equiv.),
–20 °C, 5 min, then NaN3 (1.5 equiv.), DMSO, 23 °C, 12 h, 86% for 2 steps; (e) oxalyl chloride (1.5 equiv.), DMSO (3.0 equiv.), CH2Cl2,
–78 °C; Et3N (4.0 equiv.) –78Ǟ0 °C, 1 h, 86%; (f) 20 (0.8 equiv.), AcOH (1.2 equiv.), NaCNBH3 (0.55 equiv.), MeOH, 0 °C, 2.5 h, 43%.
Scheme 3. Synthesis of protected PNA monomers 21 and 22. Reagents and conditions: (a) HBTU or TSTU (1.1 equiv.), EtiPr2N (1.3–
3.0 equiv.), 7, 10, 13, or thymine-1-acetic acid (1.1 equiv.), DMF, 23 °C, 4–7 h, 55–94%; for coupling to 17e (Azoc): 10f or 13f (1.1 equiv.),
pivaloyl chloride (1.15 equiv.), N-methylmorpholine (2ϫ2.5 equiv.), DMF, 0 °C Ǟ23 °C, 4 h; (b) LiOH (3.0–4.0 equiv.), 1,4-dioxane/H2O
or MeOH/H2O, 23 °C, 2–20 min, 37–98%; (c) TSTU (1.1 equiv.), EtiPr2N (1.3 equiv.), 7f, 10f, 13f, DMF, 23 °C, 1 h, 62–74%.
methods led to the formation of a mixed anhydride between Alloc group of the N-terminus were indeed more stable to
17f and 3 thus engendering higher-order oligomers in ad- TFA. As shown in Scheme 4, we thus prepared tetramer 25
dition to desired product 22.[18]
containing all four nucleobases, which was released from
Our first objective was to investigate the possibility of the resin by using 0.5% TFA in CH2Cl2 to afford fully pro-
preparing PNA oligomers for segment couplings.[19,20] Al- tected oligomer 26. The oligomer was then coupled to Rink
though we had previously used 2-chlorotrityl resin to load resin and engaged in two iterative cycles of deprotection/
PNA monomers bearing an Fmoc group onto the N-ter- coupling by using DIC/HOBt activation to obtain a do-
minus and Bhoc groups on the nucleobases,[11] this system decamer. The kinetics of the couplings with the use of tetra-
suffered from two limitations: first, the basic conditions re- mer 26 were found to be significantly slower than the corre-
quired for the Fmoc deprotection led to significant cleavage sponding reaction with monomers 21 requiring at least 12 h
due to cyclization (piperazinone formation), and second, to go to completion. Considering that an excess of mono-
the Bhoc group was not compatible with 1% TFA cleavage mer is required for the preparation of tetramer 26, which is
of the 2-chlorotrityl resin and required acetic acid cleavage, in turn used in excess (Ͼ4 equiv.) in the subsequent coup-
which was difficult to remove. We thus first investigated the lings, the synthesis of oligomers by segment coupling was
Cl-Bhoc group by reasoning that the electron-withdrawing not deemed attractive for our applications.
nature of the chlorine substituents would render this group
To explore further protecting groups for the terminal ni-
more acid stable. To our gratification, we found that mono- trogen atom, we were drawn to the Teoc group for its pre-
mers 21 bearing a Cl-Bhoc group of the nucleobase and an sumed compatibility to acid-labile groups and the Alloc or
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S. Pothukanuri, Z. Pianowski, N. Winssinger
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Scheme 4. Synthesis of protected PNA oligomers for segment coup-
ling. Reagents and conditions: (a) 21 (2.0 equiv.), EtiPr2N
(2.0 equiv.), DMF, 23 °C, 4 h; (b) Pd(Ph3)2Cl2 (0.1 equiv.), Me3SiN3
(10 equiv.), Bu3SnH (10 equiv.), CH2Cl2, DMF, 23 °C, 1 h; (c) 21
(4.0 equiv.), HBTU (3.5 equiv.), EtiPr2N (8.0 equiv.), NMP, 23 °C,
4 h; (d) TFA (0.5%) in CH2Cl2, 23 °C, 4 ϫ10 min.
Scheme 5. Deprotection of Teoc-protected PNA monomers 27 and
29. Reagents and conditions: (a) n-butylamine (1.5 equiv.), HBTU
(1.5 equiv.), EtiPr2N (3.0 equiv.), DMF, 23 °C, 4 h, 67%; (b) 1 
TBAF in DMSO or DMF, 23 °C, 10 min.
azide-based protecting groups. To evaluate the kinetics of
deprotection, monomer 21 (Scheme 5) bearing a Teoc
group on the terminal nitrogen atom and a Cl-Bhoc group
on the exocyclic nitrogen atom of cytosine was coupled to
butylamine thus affording compound 27. This compound
was found to undergo smooth deprotection in 10 min with
no evidence of acyl group migration by using 1  TBAF in
DMF or DMSO. Encouraged by this result, the reaction
was repeated with the polymer-bound equivalent (29); how-
ever, the yield of the reaction was disappointingly low
(Ͻ10% deprotection after 30 min). This result was surpris-
ing considering the successful prior use of silyl ethyl linker
for carboxylic acid in solid-phase synthesis.[21,22] We as-
cribed the difference in reactivity between the solution-
phase result (27Ǟ28) and the solid-phase result (29Ǟ30) to
the hydrophobic environment of the polystyrene resin and
investigated the reaction by using different solvents (DMF,
DMSO, and DMF/MeOH) and resins (NovasynTGR: a
PEG-polystyrene resin and PEGA: a PEG-polyamide
resin);[23] however, we were unable to obtain satisfactory
results.
Figure 2. Stability of protected cytosines 6a–f toward TFA in
CH2Cl2.
Scheme 6. Deprotection of 4-N3Cbz-protected PNA monomer 31. Reagents and conditions: (a) 1  PMe3, THF/H2O (9:1) or 1 
PBu3,THF/H2O (9:1), 23 °C, 15 min; (b) decomposition of the azaylide (see conditions above).
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Expanding the Scope and Orthogonality of PNA Synthesis
Scheme 7. Orthogonal deprotection of Mtt and 4-N3Cbz. Reagents and conditions: (a) piperidine/DMF (1:4), 23 °C, 5 min; (b) 21
(4.0 equiv.), HBTU (3.5 equiv.), EtiPr2N (8.0 equiv.), NMP, 23 °C, 4 h; (c) 1  PMe3, THF/H2O (9:1), 23 °C, 15 min; 1  Cl3CO2H, THF/
H2O (9:1), 23 °C, 4 h; (d) Ac2O (0.2 ), 2,6-lutidine (0.2 ), DMF, 23 °C, 5 min; (e) TFA (1%), CH2Cl2, 23 °C, 30 min; (f) Ac2O (0.2 ),
2,6-lutidine (0.2 ), DMF, 23 °C, 5 min.
Scheme 8. PNA synthesis by using Boc/Fmoc protected monomers and their orthogonality to silyl ethers (TBS). Reagents and conditions:
(a) piperidine/DMF (1:4), 23 °C, 5 min; (b) 22 (4.0 equiv.) or Fmoc-Lys(Boc)-OH (4.0 equiv.), HBTU (3.5 equiv.), EtiPr2N (4.0 equiv.),
2,6-lutidine (6.0 equiv.), NMP, 23 °C, 30 min; (c) TFA (1%), CH2Cl2, 23 °C, 30 min; (d) 40 (4.0 equiv.), HBTU (3.5 equiv.), EtiPr2N
(4.0 equiv.), 2,6-lutidine (6.0 equiv.), NMP, 23 °C, 12 h.
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Inspired by the mildness of azide reduction coupled to coupled to azidocoumarin 40[25] thus establishing fully or-
the benefits of carbamate protecting groups, we investigated thogonal deprotection conditions. It is instructive to note
the suitability of the 4-N3Cbz group.[24] Although we pre- that the same compound was previously prepared by using
viously reported the preparation of azidoPNA mono- commercially available PNA monomers bearing Fmoc/
mers[10] (PNA bearing an azide at the N-terminus rather Bhoc protecting groups; however, the Fmoc group had to
than a carbamate protecting group), rigorous application of be exchanged prior to TBAF deprotection of the silyl ether,
the deprotection protocol had to be utilized to avoid chain as that protecting group combination was not orthogonal
termination due to cyclization of the iminophosphorane to to silyl ether deprotection.
a dihydroimidazole. This problem was overcome by acylat-
On the basis of the decomposition of iminophosphorane
ing directly the iminophosphorane product obtained; how- 32 and unsuitability of the Teoc group for our purpose, we
ever, this methodology is not well suited for automated syn- speculate that the 4-OTBSCbz group may offer a useful
thesis, which thus limits the applicability of azidoPNA. To alternative fluoride-labile protecting group. Resin 42
evaluate the 4-N3Cbz group, Rink resin 31 (Scheme 6) bear- (Scheme 9) loaded with a protected PNA monomer (Boc/4-
ing a protected PNA monomer (Cl-Bhoc/4-N3Cbz) was OTBSCbz) was exposed to TBAF, which thus resulted in
treated either with tributyl phosphane or trimethyl phos- complete deprotection within minutes. However, desired
phane in THF/H2O (9:1) thus affording iminophosphorane product 43 was contaminated with Ͼ50% of product 44
32, which decomposed into amine 30. In our hands, the presumably stemming from the reaction of 43 with the qui-
decomposition of 32 was sluggish and required an acid to none methide generated under the deprotection conditions.
proceed within a reasonable time frame.
Attempts to suppress this side reaction with the addition of
The most practical reactions conditions were the use of reducing agents or cation scavengers such as Et3SiH, m-
trichloroacetic acid at room temperature for three hours to cresol, Me2S, or amines only led to marginal improvements.
obtain complete deprotection. In order to assess the orthog-
onality of the 4-N3Cbz group with very acid-labile groups
such as amino-protected methyl trityl (Mtt), resin 33 was
coupled to 4-N3Cbz-protected monomer 21 (Scheme 7) and
engaged in an orthogonal deprotection of either the Mtt
(1% TFA) or 4-N3Cbz (PMe3; Cl3CO2H) group followed by
acylation to obtain 35 and 36, respectively, thus establishing
mutual orthogonality of these two groups. However, cau-
tion had to be employed in the deprotection of the Mtt
group, as prolonged exposure to 1% TFA led to Cl-Bhoc
deprotection. This latter fact incited us to reinvestigate
more acid-stable groups for the nucleobases. As shown in
Figure 2, the stability of cytosine 6a–f bearing Bhoc, Cl-
Bhoc, F-Bhoc, 4-OMeCbz, Teoc, and Boc groups, respec-
tively, were compared when exposed to 1% TFA and 5%
TFA for 20 min. Cytosine was used for this experiment, as
it is the most susceptible nucleobase to acid-mediated de-
protection. Aside from the Bhoc group, all other groups
were stable to 1% TFA; the Boc group was the most stable
and showed minimal deprotection even with 5% TFA after
20 min. On the basis of the ease of removing the Boc by-
product after deprotection and the fact that adenine 9f and
guanine 12f were even more resistant to acid, the Boc group
was deemed most attractive for our purpose and F-Bhoc-,
4OMeCbz-, and Treoc-protected nucleobases were not fur-
ther investigated.
Having access to PNA monomers bearing more acid-re-
sistant protecting groups on the nucleobases, we asked
whether it would be possible to deprotect a silyl ether under
acidic conditions in the presence of an Fmoc-protected
PNA oligomer. The basicity of fluoride in TBAF makes the
fluoride-based deprotection of silyl ether incompatible with
Fmoc groups. Thus, resin 38 bearing an N-(2-aminoethyl)
Scheme 9. Deprotection of 4-OTBSCbz protected PNA 42. Rea-
gents and conditions: 1  TBAF in THF, 23 °C, 5 min.
serine PNA monomer was engaged in a cycle of nine
We recently reported the use of the Azoc group in the
iterative deprotection/coupling to obtain decamer 39 context of peptide and carbohydrate chemistry and showed
(Scheme 8). The TBS group was removed quantitatively by it to be mutually orthogonal to both the Mtt and Fmoc
using 1% TFA, and the resulting free hydroxy group was groups thus allowing the parallel synthesis in three dimen-
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Expanding the Scope and Orthogonality of PNA Synthesis
sions.[17] Encouraged by its fast deprotection under very additional dimension of orthogonality, which may be useful
mild conditions,[26] we investigated its utility for PNA syn- to introduce reporter groups such as fluorophores. The ra-
thesis. As shown in Scheme 10, the deprotection of poly- pid deprotection of the Azoc group and the mildness of the
mer-bound intermediate 45 was extremely fast and contrar- reagents makes it compatible with the fast cycles of auto-
ily to the aforementioned 4-N3Cbz group, did not require mated PNA synthesizers and rivals the efficiency of Fmoc-
an acid for the iminophosphorane decomposition. Azoc- based synthesis.[27] The most convenient combination of
protected monomers were found to perform with equal effi- protecting groups was found to be Boc-protected nucleo-
ciency in PNA synthesis to Fmoc-protected monomers and bases with either Azoc or Fmoc protecting groups for the
are compatible with automated synthesis.
terminal nitrogen atom. The added stability of the Boc
group relative to that of the Cl-Bhoc or F-Bhoc group
makes reiterative deprotection of Mtt or other acid-labile
groups reliable. Although the Alloc group can be removed
in the presence of an Azoc or Fmoc group, reiterative de-
protection of the Alloc group with tin hydride and palla-
dium tetrakis leads to the precipitation of palladium, which
can catalyze the decomposition of Bu3SnH into H2 thus
leading to partial reduction of the Alloc group to the propyl
carbamate or Azoc deprotection. Although this side reac-
tion is marginal, it does decrease the efficiency of a reitera-
tive process.
Experimental Section
Full experimental details and characterization data for compounds
outlined in this work are available in the Supporting Information.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures and spectroscopic data for nucleo-
bases 6, 7, 9, 10, 12, and 13 and PNA monomers 21 and 22; chro-
matograms and MALDI spectra of oligomers 41 and 49.
Acknowledgments
The French Ministry of Research and Education is gratefully
acknowledged for a fellowship (MRT to Z. P.). This project was
partly supported by grant from the Agence National de la Recher-
che (ANR) and Human Frontier Science Program (HFSP).
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Received: February 5, 2008
Published Online: May 9, 2008
3148
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Eur. J. Org. Chem. 2008, 3141–3148