A Fmoc-based submonomeric strategy for the solid phase synthesis of optically pure chiral PNAs

Article abstract of DOI:10.1016/j.tetlet.2008.05.114

A new submonomeric approach for the Fmoc solid phase synthesis of chiral PNAs is reported here. The design and synthesis of a new d-lysine-based Fmoc submonomer obtained by replacing the nucleobase residue with an Alloc group, compatible with Fmoc chemistry, is described starting from d-lysine and Fmoc-aminoacetaldehyde. The desired submonomer was obtained in high yield with no racemization. The conditions for synthesizing a chiral PNA dimer on solid support by a submonomeric approach have been optimized, allowing to obtain the desired PNA with a very high optical purity.

Full text of DOI:10.1016/j.tetlet.2008.05.114

Tetrahedron Letters 49 (2008) 4958–4961
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A Fmoc-based submonomeric strategy for the solid phase synthesis
of optically pure chiral PNAs
*
Tullia Tedeschi, Stefano Sforza , Francesca Maffei, Roberto Corradini, Rosangela Marchelli
Dipartimento di Chimica Organica e Industriale, Università di Parma, Viale G.P. Usberti, 17a, Parma 43100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new submonomeric approach for the Fmoc solid phase synthesis of chiral PNAs is reported here. The
Received 14 April 2008
Revised 20 May 2008
Accepted 23 May 2008
Available online 17 June 2008
design and synthesis of a new
residue with an Alloc group, compatible with Fmoc chemistry, is described starting from
Fmoc-aminoacetaldehyde. The desired submonomer was obtained in high yield with no racemization.
The conditions for synthesizing a chiral PNA dimer on solid support by a submonomeric approach have
been optimized, allowing to obtain the desired PNA with a very high optical purity.
Ó 2008 Elsevier Ltd. All rights reserved.
D
-lysine-based Fmoc submonomer obtained by replacing the nucleobase
-lysine and
D
Keywords:
Chiral PNAs
Submonomeric strategy
Optical purity
Fmoc strategy
Peptide nucleic acids (PNAs) are well-known DNA mimics intro-
duced by Nielsen and co-workers in 1991.1 Their structure is based
on a neutral and achiral pseudopeptide backbone based on N-(2-
aminoethyl)glycyl units, with nucleobases linked to the secondary
amines by carboxymethylene linkers. PNAs are able to bind com-
plementary DNA/RNA sequences through standard Watson–Crick
hydrogen bonds with high sequence specificity and affinity.2 For
their outstanding hybridization properties, together with their
high chemical and biological stability, PNAs have found many
applications in different areas, including bioorganic chemistry,
drug discovery, molecular biology, diagnostics, prebiotic evolution
and material science.3
Base
O
R''
1'
O
6
2
4
N
*
3
1
N
H
5
n
*
R'
R '= R'' = H: standard achiral PNA
R' = amino acid side chain, R''=H: chiral 2-substituted PNA
R' = H, R'' = amino acid side chain: chiral 5-substituted PNA
R' = R''= amino acid side chain: chiral 2,5-substituted PNA
Figure 1. Schematic representation of achiral and chiral PNAs.
Many examples of modified PNAs have been developed by
introducing functional groups with different charge, polarity and
flexibility.4,5 Amongst modified PNAs, chiral amino acid-based ana-
logues substituted in position 2, position 5 or both (Fig. 1) have re-
cently gained increasing attention, due to their peculiar DNA
binding abilities easily tunable according to the different configu-
rations,6,7 to the new properties induced by the aminoacidic resi-
dues8,9 and to the possibility to use amino acid side chains in
order to link new functionalities.10,11
Since the performances of chiral PNAs in terms of binding spec-
ificity and selectivity are strongly affected by the configurations of
the stereogenic centers, their optical purity is a primary issue,
although often neglected. In particular, PNA synthesis of chiral
derivatives substituted in position 2 can be strongly affected by
racemization, as demonstrated by a GC method developed by our
group.12 In order to maintain a very high optical purity, a Boc-
based ‘submonomeric strategy’ for the solid phase synthesis of
these oligomers was developed:13 according to this strategy, the
Boc-protected chiral backbone, carrying a Fmoc group on the sec-
ondary amine in place of the nucleobase acetyl moiety, was cou-
pled to the PNA growing chain on the resin, followed by Fmoc
deprotection and resin coupling of the nucleobase acetic acid.
The assembly of the full chiral PNA residue directly on resin
allowed for a fast and almost racemization-free synthesis of chiral
PNAs. Although successful, this protocol has the main drawback of
using the harsh synthetic conditions of the Boc-chemistry. Since
the conjugation of PNAs with fluorophores and other labelling mole-
cules, which are often labile at strong acidic conditions, is becom-
ing of great interest, the use of a milder chemistry, such as in Fmoc-
based strategies, should be advisable. Recently, Seitz and co-work-
ers14 have proposed a submonomeric approach in the synthesis of
achiral PNAs as FIT (Force Intercalation of Thiazole orange) probes.
* Corresponding author. Tel.: +39 0521905406; fax: +39 0521905472.
E-mail address: stefano.sforza@unipr.it (S. Sforza).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.114

T. Tedeschi et al. / Tetrahedron Letters 49 (2008) 4958–4961
4959
Hence, in this Letter we propose for the first time a new Fmoc-
O
O
H2N
+
based submonomeric strategy for the synthesis of optically pure
chiral PNAs. We report the design and the synthesis of a new
Fmoc-compatible submonomeric unit carrying a lysine side chain
at the carbon 2. This new submonomer has been used in the syn-
thesis of a chiral PNA dimer on solid phase support by Fmoc proto-
col. The preservation of the optical purity all along the synthetic
steps was also demonstrated.
H
OH
O
N
H
O
1
HN
O
i
O
O
O
O
O
Synthesis of the Fmoc-submonomeric unit based on
2D-Lysine
O
O
H
N
N
backbone: In order to obtain a chiral PNA submonomer Fmoc pro-
tected at the N-terminus, Fmoc-N-aminoacetaldehyde 1 was first
synhtesized. Its synthesis has been reported in the literature,
although with low yields, by reduction with LiAlH4 of the Fmoc
glycine Weinreb amide.15 We tried this approach in comparison
with a new one based on the oxidation of the Fmoc-N-( )-3-ami-
no-1,2-propandiol with KIO4 (Scheme 1). In both strategies the first
steps, the synthesis of the Weinreb amide from the Fmoc-glycine
in one case and the introduction of the Fmoc group on the ( )-3-
amino-1,2-propandiol in the other case, were obtained with quite
high yields. In contrast, the reduction with LiAlH4 of the Weinreb
amide was not very efficient, whereas the diol oxidation by perio-
date was almost quantitative, making the second route preferable.
O
N
H
OH
O
N
H
OH
ii
HN
3
O
HN
O
2
O
O
Scheme 2. Reagents and conditions: (i) 1 equiv NaBH3CN, 1.1 equiv CH3COOH,
CH3OH, : 53%; (ii) 2 equiv Alloc–Cl, 1.5 equiv DIPEA THF, : 91%.
g
g
group introduction was initially carried out by following the same
conditions used for the introduction of Fmoc group on Boc-lysine
backbone: Alloc–Cl/DIEA in DCM with bis(trimethylsilyl)acetamide
(BTSA). BTSA was added in order to increase the reagent solubility
by formation of a transient silyl ester, which was also previously
found essential in order to avoid racemization.13 Unfortunately,
the reaction was totally unsuccesful by this strategy. Also the sub-
stitution of Alloc–Cl with Alloc anydride did not improve the
yields. As a further attempt, BTSA was excluded from the reaction
mixture and DCM was replaced with a more polar solvent (THF). In
these conditions the reaction was successful and the submonomer
3 was obtained with high yields (90%) (Scheme 2).
Then, the 2
nation of 1 with H-
D
-lysine backbone 2 was obtained by reductive ami-
-Lys(Boc)-OH. Although this reaction is usually
D
performed with the carboxyl function protected as an ester,13 in
this case the reaction was performed directly on the amino acid
residue without protecting the carboxyl function (Scheme 2). Con-
sistent with studies performed by Gilon and co-workers16 on other
amino acid derivatives, this procedure gave good yields and, as a
further advantage, the product was easily purified by direct precip-
itation from the reaction mixture.
In order to obtain a suitable submonomer to be used in a solid
phase Fmoc-PNA synthesis, it appeared necessary to have a pro-
tecting group on the secondary amine, in place of the nucleobase
acetyl moiety, which could be cleaved without affecting neither
the temporary Fmoc group at the N-terminus nor the orthogonal
Boc-protecting group used for the lysine side chain. Since the pres-
ence of a carbamate as protecting group in this position was found
to be essential in order to maintain a high optical purity during
coupling,13 the allyloxycarbonyl (Alloc) group was chosen. This
group is removed by nucleophilic cleavage in the presence of pal-
ladium, being thus orthogonal both to Fmoc and Boc-groups, and it
has been already tested on the achiral PNAs chemistry.14 The Alloc
In order to check if the lack of the in situ protection of the car-
boxylic function had caused racemization, a chiral analysis was
performed by using the chiral GC method previously developed.12
For both 2 and 3 an ee of 99% was found, indicating that this syn-
thetic procedure does not affect the optical purity of the lysine
backbone.
All the compounds described here were characterized by 1H, 13
C
NMR and ESI mass spectrometry. The characterization of com-
pounds 1, 2 and 3 is reported.17
Solid phase synthesis of a chiral PNA dimer based on 2D-lysine by
Fmoc submonomeric strategy: The submonomer synthesized above
was used for the solid phase synthesis of the chiral PNA dimer 4
by Fmoc-based submonomeric strategy (Fig. 2).
Starting from the resin preloaded with an achiral T PNA mono-
mer (Fmoc-T-Linker-AM Champion resin, 0.17 mmol/g), the
submonomer 3 was successfully inserted by manual coupling
using a standard HBTU/DIEA protocol.13
OH
OH
O
OH
H2N
O
N
H
O
iii
i
O
N
O
OH
OH
O
N
O
N
O
N
O
N
H
O
H
NH
NH
N
O
O
O
O
O
ii
iv
O
O
N
H3N+
N
O
NH2
H
H
O
N
H
O
4
1
NH3+
Scheme 1. Reagents and conditions: (i) 0.97 equiv N-methyl-N-mehoxy amine.
HCl, HBTU, DIEA, DMF, : 98%; (ii) 4.6 equiv LiAlH4 (1 M in THF), THF, : 30%; (iii)
FmocOSu, Na2CO3, 1,4-dioxane, : 80%; (iv) KIO4, H2O/acetone, : 99%.
g
g
g
g
Figure 2. Chiral PNA dimer H–T(D-Lys)T–NH2 4.

4960
T. Tedeschi et al. / Tetrahedron Letters 49 (2008) 4958–4961
O
N
NH
O
O
O
O
N
Peg-PS
resin
O
N
N
H
H
20% piperidine/NMP
O
NH
N
O
Figure 3. ESI-MS spectrum of the purified chiral PNA dimer 4. m/z: 621.4: [M+H+]+,
455.3: [M–ThyCH2CO++2H+]+, 311.1: [M+2H+]2+
ThyCH2CO+.
,
302.7: [MÀNH3+2H+]2+
, 167.0:
O
O
Peg-PS
resin
N
H2N
N
H
3
HBTU,DIEA,NMP
a
The next step was the deprotection of the N -Alloc protecting
group. The Alloc removal is usually done through the allyl group
transfer via nucleophylic substitution, which takes place in the
presence of a palladium complex. According to the literature,18
the dimethyl amino borane complex was used as cleaving reagent,
by using the same conditions reported by Seitz and co-workers14
(0.1 equiv [Pd(PPh3)4], 8 equiv Me2NHBH3 in DCM at room temper-
ature for 40 min). After cleavage of the product from the resin, the
reaction completion was monitored by HPLC/ESI-MS analysis.
Unfortunately, these conditions were not sufficient to completely
cleave the Alloc, even after long reaction times, very likely for
the high steric hindrance of the protected amino acid side chain
nearby. Thus, the Alloc deprotection reaction was performed by
increasing the amounts of [Pd(PPh3)4] and Me2NHBH3. Complete
removal of the protecting group was finally achieved by using a
DCM solution containing 0.2 equiv of [Pd(PPh3)4] and 20 equiv of
Me2NHBH3 and by repeating the reaction two times for 1 h each.
For the introduction of the thymine acetic acid, the same cou-
pling reaction conditions already used for the Boc submonomeric
strategy,13 which are consistent with the Fmoc group, were ap-
plied, by using DIC/DhBTOH in NMP. After removal of the terminal
Fmoc group by a 20% solution of piperidine in NMP, the chiral PNA
dimer 4 was cleaved from the solid support with a TFA/m-cre-
sol = 9:1 mixture (Scheme 3).
O
NH
N
O
O
O
O
O
Peg-PS
resin
O
O
N
N
O
N
N
N
H
H
H
HN
O
O
MeNHBH3 20eq,
Pd(PPh3)4 0.2 eq
O
NH
N
O
O
O
O
O
Peg-PS
resin
H
N
N
O
N
N
N
H
H
H
The crude product was obtained after precipitation with diethyl
ether (yield 75% as determined by HPLC/MS). The chiral PNA dimer
4 was purified by semipreparative HPLC and characterized by ESI-
MS (Fig. 3). Its optical purity was checked by chiral GC:12 an out-
standing ee of 98% was found.
From these results it is clear that the new Fmoc submonomeric
strategy reported here is fully suitable for the obtainment of PNAs
incorporating optically pure chiral residues: the enantiomeric ex-
cess observed is higher than that obtained by standard PNA solid
phase synthesis;19 the preparation of the submonomer can be
made in fewer synthetic steps than the previously reported Boc
protocol, and the protocol can be used for conjugating PNAs with
fluorophores or other labelling molecules labile in strong acidic
conditions.
Thy-CH2COOH
DhBTOH, DIC
HN
O
O
O
O
NH
NH
N O
O
N O
O
O
O
O
Peg-PS
resin
N
N
O
N
N
N
H
H
H
HN
O
20% piperidine/NMP
TFA/m-cresol 9:1
O
Acknowledgement
4
Financial support from Italian Ministry of Instruction,
University and Research (MIUR) through Projects of Relevant
National Interest (PRIN 2005, Grant No. 200503874) is gratefully
acknowledged.
Scheme 3. Scheme of the submonomeric cycle for the insertion of
monomer on a PNA chain on solid phase by Fmoc protocol.
a chiral

T. Tedeschi et al. / Tetrahedron Letters 49 (2008) 4958–4961
4961
17. Compound 1: 1H NMR (300 MHz, CDCl3): d 9.6 (s, 1H, C–H aldehyde), 7.7 (d,
2H, J = 7.5, C–H aromatic Fmoc), 7.6 (d, 2H, J = 7.1, C–H aromatic Fmoc), 7.4–7.2
(m, 4H, C–H aromatic Fmoc), 5.5 (br s, 1H, N–H carbamate), 4.4 (d, 2H, J = 6.7
CH2 Fmoc), 4.2 (t, 1H, J = 6.7, C–H Fmoc), 4.1 (d, 2H, J = 4.5 CH2 glycine). 13C
NMR (75 MHz,CDCl3): d 196.5, 156.2, 143.7, 141.3, 127.7, 127, 124.9, 119.9,
67.1, 51.6, 47. ESI (CH3OH, positive ions) calcd m/z: 336.1 (M+CH3OH+Na+),
352.1(M+CH3OH+K+), found: 336.3 (M+CH3OH+Na+), 352.3(M+CH3OH+K+).
Compound 2: 1H NMR (300 MHz, DMSO-d6): d 7.9–7.8 (m, 2H, aromatic Fmoc),
7.7–7.6 (m, 2H, aromatic Fmoc), 7.4–7.3(m, 4H, aromatic Fmoc), 6.7 (br s, 1H,
N–H carbamate), 6.3 (br s, 1H, N–H carbamate), 4.3–4.2 (m, 3H, CH+CH2 Fmoc),
3.2–3.1 (m, 3H, C(2)–H + C(5)–H2), 2.9–2.7 (m, 4H, C(4)–H2 + CH2 lysine side
chain), 1.5–1.3 (m, 15H, CH2 lysine side chain + CH3 Boc). 13C NMR (75 MHz,
DMSO-d6): d 72.2, 156, 155.3, 143.7, 142.2, 128.7, 127.4, 127.1, 126.9, 124.9,
121.2, 120, 119.8, 77.2, 66, 61.7, 46.5, 40.1, 38.1, 30.7, 29.3, 28.1, 22.3, 21.4. ESI
(CH3OH, positive ions) calcd m/z: 512.3 (MH+), 534.3 (MNa+), 549.3 (MK+),
found: 512.5, 534.5, 549.5. Ee (chiral GC–MS according to the method reported
in the literature12): 99%.
References and notes
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254,
1497.
2. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D.
A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365,
566.
3. Lundin, K. E.; Good, L.; Stromberg, R.; Gaslund, A.; Smith, C. I. E. Adv. Genet.
2006, 56, 1–51.
4. Kumar, V. A.; Ganesh, K. N. Acc. Chem. Res. 2005, 38, 404–412.
5. Corradini, R.; Sforza, S.; Tedeschi, T.; Totsingan, F.; Marchelli, R. Curr. Top. Med.
Chem. 2007, 7, 681–694.
6. Sforza, S.; Haaima, G.; Marchelli, R.; Nielsen, P. E. Eur. J. Org. Chem. 1999, 197–
204.
7. Sforza, S.; Tedeschi, T.; Corradini, R.; Marchelli, R. Eur. J. Org. Chem. 2007, 5879–
5885.
8. Dragulescu-Andrasi, A.; Rapireddy, S.; He, G.; Bhattacharya, B.; Hyldig-Nielsen,
J.J.; Zon, G.; Ly, D.H.; J. Am. Chem. Soc., 2006, 128, 50, 16104.
9. Rapireddy, S.; He, G.; Roy, S.; Armitage B.A.; Ly, D.H.; J. Am. Chem. Soc., 2007,
129, 50, 15596.
10. Englud, E.; Appella, D.H.; Org. Lett., 2005, 7, 16, 3465.
11. Englund, E. A.; Appella, D. H. Angew. Chem., Int. Ed. 2007.
12. Corradini, R.; Di Silvestro, G.; Sforza, S.; Palla, G.; Dossena, A.; Nielsen, P. E.;
Marchelli, R. Tetrahedron: Asymmetry 1999, 10, 2063.
13. Sforza, S.; Tedeschi, T.; Ciavardelli, D.; Corradini, R.; Dossena, A.; Marchelli, R.
Eur. J. Org. Chem. 2003, 1056.
14. Jarikote, D. V.; Kohler, O.; Socher, E.; Seitz, O. Eur. J. Org. Chem. 2005, 3187–
3195.
15. Baghavanthy, S. B.; Gallazzi, F.; Jia, F.; Lewis, M. R. Bioconjugate Chem. 2006, 17,
551–558.
16. Bitan, G.; Muller, D.; Kasher, R.; Gluhov, E. V.; Gilon, C. J. Chem. Soc., Perkin
Trans. 1 1997, 1501–1510.
Compound 3: 1H NMR (300 MHz, DMSO-d6): d 10.3: d 10.3 (br s, 1H, CO2–H
carboxylic acid), 7.8 (d, 2H J = 7.3, aromatic Fmoc), 7.6 (d, 2H, J = 7.3 aromatic
Fmoc), 7.4 (t, 2H, J = 7.3, aromatic Fmoc), 7.3 (t, 2H, J = 7.3, aromatic Fmoc),
5.9–5.8 (m, 1H, C–H Alloc group), 5.3–5.2 (m, 2H, CH2 alloc group), 4.6–4.5 (m,
2H, CH2 alloc group), 4.3–4.2 (m, 3H, CH + CH2 Fmoc), 3.9 (br s, C(2)–H), 3.4–
3.3 (m, 2H, C(5)–H2), 3.2–3.1 (m, 2H, C(4)–H2), 2.9–2.8 (m, 2H CH2 lysine side
chain), 1.4–1.2 (m, 15H, CH2 lysine side chian + CH3 Boc). 13C NMR (75 MHz,
DMSO-d6): d 72.6, 155.9, 155.4, 155.2, 149.6, 143.7, 140.6, 132.9, 127.4, 126.8,
124.9, 119.9, 116.5, 77.1, 65.2, 61.6, 59.6, 46.5, 45.4, 41.4, 39.5, 29.1, 28.3, 28.1,
23.3. ESI (CH3OH, positive ions) calcd m/z: 618.3(MNa+), 633.3(MK+), found:
618.5, 633.5. Ee (chiral GC–MS according to the method reported in the
literature12): 99%.
18. Gomez-Martinez, P.; Dessolin, M.; Guibè, F.; Albericio, F. J. Chem. Soc., Perkin
Trans. 1 1999, 2871–2874.
19. Tedeschi, T.; Corradini, R.; Marchelli, R.; Pushl, A.; Nielsen, P. E. Tetrahedron:
Asymmetry 2002, 13, 1629.