Synthesis and properties of novel pyrrolidinyl PNA carrying β-amino acid spacers

Article abstract of DOI:10.1016/S0040-4039(01)01020-6

Novel pyrrolidinyl peptide nucleic acids comprising alternate sequences of nucleobase-modified d-proline and β-amino acid spacers selected from L-aminopyrrolidine-2-carboxylic acid, D-aminopyrrolidine-2-carboxylic acid, (1R,2S)-2-aminocyclopentane carboxylic acid and β-alanine were synthesized using solid phase methodology. Gel-binding shift assay revealed that only the homothymine PNA decamer bearing D-aminopyrrolidine-2-carboxylic acid spacer binds with (dA)₁₀.

Full text of DOI:10.1016/S0040-4039(01)01020-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5533–5536
Synthesis and properties of novel pyrrolidinyl PNA carrying
b-amino acid spacers
Tirayut Vilaivan,a,* Chaturong Suparpprom,a Pongchai Harnyuttanakornb and Gordon Lowec
aOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,
Patumwan, Bangkok 10330, Thailand
bDepartment of Biology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
cDyson Perrins Laboratory, Department of Chemistry, Oxford University, South Parks Road, Oxford, OX1 3QY, UK
Received 24 April 2001; accepted 8 June 2001
Abstract—Novel pyrrolidinyl peptide nucleic acids comprising alternate sequences of nucleobase-modified
D
-proline and b-amino
acid spacers selected from -aminopyrrolidine-2-carboxylic acid, -aminopyrrolidine-2-carboxylic acid, (1R,2S)-2-aminocyclopen-
L
D
tane carboxylic acid and b-alanine were synthesized using solid phase methodology. Gel-binding shift assay revealed that only the
homothymine PNA decamer bearing
All rights reserved.
D-aminopyrrolidine-2-carboxylic acid spacer binds with (dA)10. © 2001 Elsevier Science Ltd.
Nielsen and his collaborators have shown that oligonu-
cleotide analogues in which the sugar-phosphate back-
bone is replaced by a poly(N-aminoethylglycine) with
the nucleobases attached through a methylenecarbonyl
group at the glycine nitrogen (1) strongly hybridize with
complementary oligonucleotides.1 The strong bonding
of these polyamide nucleic acids (PNAs) was attributed,
in part, to the lack of negative charge on the PNA.2 In
natural oligonucleotides or their phosphorothioate ana-
logues, electrostatic repulsion between the negatively
charged strands offsets the attractive forces from
hydrogen bonding and p-stacking of the bases. PNAs
have also been shown to be more selective in recogniz-
ing their complementary sequences, the greater discrim-
ination against mismatched sequences arising from the
greater binding contribution from each nucleobase. We
have recently developed this concept further and gener-
ated a new class of oligonucleotide analogues derived
from the pyrrolidine core structure (2).3–5 A number of
related pyrrolidine PNAs have been reported recently,
many of which showed binding to their complementary
oligonucleotides.6–11
We believe it should be possible to enhance further the
binding of these PNAs to complementary oligonucleo-
tides by replacing the glycine residue in (2) with an
alternative spacer with an appropriate conformational
rigidity. Molecular modeling suggested that if the
glycine spacer in (2) is replaced by a b-amino acid in
which the dihedral angle between the amino group and
the carboxylic acid is close to 0°, a very favorable
geometry for hybridization to the complementary
Keywords: nucleic acids; DNA; mimetics; b-amino acids.
* Corresponding author. Tel.: +66-2-218-4961; fax: 66-2-254-1309; e-mail: vtirayut@chula.ac.th
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01020-6

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T. Vilai6an et al. / Tetrahedron Letters 42 (2001) 5533–5536
oligonucleotide should result.12 Here we report the syn-
thesis and binding studies of a series of novel pyrro-
lidinyl PNAs bearing various b-amino acid spacers (3).
aminopropyl-carbodiimide·HCl (EDC·HCl) mediated
coupling with commercially available Fmoc-b-alanine.
The dipeptide was treated with 4 M HCl in dioxane to
deprotect the diphenylmethyl ester followed by treat-
ment with pentafluorophenyl trifluoroacetate/DIEA to
give the activated dipeptide (7) in 43% overall yield
(Scheme 1).
The selected spacers include
boxylic acid ( -Apc),
acid ( -Apc), (1R,2S)-2-aminocyclopentane carboxylic
acid (
L
-aminopyrrolidine-2-car-
L
D-aminopyrrolidine-2-carboxylic
D
L
-Acpc) and b-alanine (b-ala). Initially four
homothymine PNA decamers 3a-T10, 3b-T10, 3c-T10 and
3d-T10 were selected as synthetic targets.
Oligomerization of these building blocks was performed
on Novasyn TGR resin (2.5 mmol scale for PNA 3a–c
and 5 mmol scale for PNA 3d) as previously described.17
Lysine amide was included at the C-temini of all PNA
for comparison with previous PNA in this series. Each
pentafluorophenyl activated monomer was attached to
the resin using 4 equivalents of the monomer and 4
equivalents of 1-hydroxy-7-azabenzotriazole (HOAt) in
DMF (60 min, single coupling). Capping (Ac2O/DIEA)
was performed after each step. After removal of the
N-Fmoc group by treatment with 20% piperidine in
DMF, the synthesis cycle was repeated until the com-
plete sequences of 3a-T10, 3b-T10, 3c-T10 and 3d-T10
were obtained. Quantitative monitoring of the dibenzo-
fulvene-piperidine adduct released during Fmoc group
deprotection showed that all coupling reactions pro-
ceeded efficiently (average coupling efficiency per cycle:
3a-T10 98.0, 3b-T10 99.2%, 3c-T10 99.8, 3d-T10 96.4%).
The crude PNAs were released from the resin by treat-
ment with trifluoroacetic acid without prior deprotec-
tion of the Fmoc group in order to use it as a
purification handle.18 After purification by reverse
phase HPLC, the Fmoc-ON PNAs were treated with
20% piperidine in DMF to give the fully deprotected
PNAs which were characterized by ESI-mass spec-
trometry (Table 1).
Syntheses of 3a-T10, 3b-T10 and 3c-T10 require the pro-
tected building blocks including Fmoc-L-aminopyrro-
lidine-2-carboxylic acid pentafluorophenyl ester (4a),
Fmoc- -aminopyrrolidine-2-carboxylic acid penta-
D
fluorophenyl ester (4b), Fmoc-(1R,2S)-2-aminocy-
clopentane carboxylic acid pentafluorophenyl ester (4c)
and Fmoc-protected PNA monomer (6). Both
-aminopyrrolidine-2-carboxylic acid were synthesized
from - and -proline via the corresponding N-nitro-
soprolines.13 Protection of
- and -aminopyrrolidine-
D- and
L
D
L
D
L
2-carboxylic acid with Fmoc-Cl followed by activation
with pentafluorophenyl trifluoroacetate/DIEA14 gave
pentafluorophenyl esters (4a) and (4b), respectively. (−)-
Cispentacin [(1R,2S)-2-aminocyclopentane carboxylic
acid]15 was similarly protected and activated to give the
pentafluorophenyl ester (4c). Boc-protected thymine
monomer (5)16 was converted to the activated Fmoc-
protected PNA monomer (6) in four steps (Scheme 1).
The PNA syntheses were performed in a stepwise fash-
ion without pre-formation of the dipeptide building
blocks. For comparison, the flexible b-alanine was also
used as spacer. In this case the dipeptide building block
was synthesised by selective deprotection of the N-Boc
group in the thymine monomer (5) by p-toluenesulfonic
acid-acetonitrile16 followed by N-ethyl-N%-dimethyl-
Scheme 1.
Table 1. ESI-MS spectra of the PNAs 3a–3d
PNA
Mr found
Mr calcd
3a-T10 3476.25, 3515.00, 3553.75
3478.69 (M), 3516.79 (M-H+K), 3554.88 (M-2H+2K)
3b-T10 3478.54, 3516.70, 3539.92, 3554.69
3478.69 (M), 3516.79 (M-H+K), 3538.77 (M-2H+Na+K), 3554.88 (M-2H+2K)
3c-T10
3469.08, 3492.56, 3507.13, 3531.48 and 3468.82 (M); 3490.80 (M-H+Na); 3506.91 (M-H+K), 3528.89 (M-2H+K+Na), 3545.01
3544.16
3d-T10 3067.00, 3089.13 and 3104.17
(M-2H+2K)
3068.16 (M); 3090.14 (M-H+Na); 3106.25 (M-H+K)

T. Vilai6an et al. / Tetrahedron Letters 42 (2001) 5533–5536
5535
aminocyclopentane carboxylic acid with the
L
-configu-
ration at the a-carbon of the spacer also did not show
evidence of binding to (dA)10. PNA 3d bearing a rela-
tively flexible b-alanine spacer, should, in principle, be
able to adopt any conformation required for binding to
its complementary DNA. The lack of binding to DNA
in this case may arise from the fact that entropy loss
during the binding process would be high. At present it
is not yet clear whether the presence of a rigid spacer
amino acid with the correct stereochemistry alone is
responsible for the unique binding behavior of b-PNA
3b or whether there is a positive contribution to the
binding resulting from attraction between the nega-
tively charged phosphate groups of DNA and the pro-
tonated pyrrolidines in 3a and 3b as well.
Figure 1. Gel hybridization experiment: Lane 1: FdA10+3d-
T10; Lane 2: FdA10+3c-T10; Lane 3: FdA10+3a-T10; Lanes 4–5:
not used; Lane 6–8: FdA10+3b-T10 1:1, 1:2, 1:5; Lane 9: not
used; Lane 10: FdA10 (negative control). Conditions: the
electrophoresis experiments were carried out on 15% poly-
acrylamide gel in 90 mM TBE buffer pH 8.3 at a constant DC
voltage of 100 V.
In conclusion, we have developed a synthetic route for
novel pyrrolidine PNAs bearing four different b-amino
acid spacers. Preliminary binding studies of these PNAs
using a gel electrophoresis technique revealed that the
structure of the amino acid spacer plays an important
role in determining the ability of these PNAs to bind to
their complementary oligonucleotide.
Preliminary binding studies of the four novel b-PNAs
to oligodeoxyribonucleotides were carried out by the
polyacrylamide gel binding shift technique using
fluorescently labelled (dA)10 [FdA10] as a probe. After a
brief incubation of 1:1 mixtures of the PNA and DNA
at 20°C, the samples were electrophoresed in 15% poly-
acrylamide gel at the same temperature. Only PNA
Acknowledgements
We acknowledge financial support from the Royal
Society of London (to T.V.), Development Grants for
New Faculty/Researchers from Chulalongkorn Univer-
sity (to T.V.) and DPST scholarship (to C.S.). We also
thank Professor S.G. Davies (Dyson Perrins Labora-
tory, Oxford) for the generous supply of (−)-cis-
pentacin, Dr. R.T. Aplin (Dyson Perrins Laboratory,
Oxford) for measuring the ESI-mass spectra and Dr.
M.S. Westwell (Dyson Perrins Laboratory, Oxford) for
molecular modeling and helpful discussion.
3b-T10 bearing
a
D-2-aminopyrrolidine-2-carboxylic
acid spacer showed positive results as evidenced by the
presence of a new slow-moving fluorescent band and
the disappearance of the fluorescent (dA)10 band (Fig.
1). Furthermore, only 3b-T10 showed observable melt-
ing with poly(dA) with a Tm value greater than 80°C at
150 mM NaCl and pH 7. Investigation of the detailed
binding properties of PNA 3b to its complementary
oligonucleotides will be published elsewhere.
The binding of b-PNAs to their complementary
oligonucleotides is remarkable since this appeared to
violate Nielsen’s ‘6+3’ principle.19 It is also of interest
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