Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): Design, synthesis and DNA/RNA binding studies

Article abstract of DOI:10.1039/b413542c

One-carbon extended conformationally constrained pyrrolidine PNA monomer (bepPNA) has been synthesized, incorporated into PNA sequences at predefined positions, and showed selective RNA binding properties.

Full text of DOI:10.1039/b413542c

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): design,
synthesis and DNA/RNA binding studies{
T. Govindaraju and Vaijayanti A. Kumar*
Received (in Cambridge, UK) 3rd September 2004, Accepted 12th October 2004
First published as an Advance Article on the web 2nd December 2004
DOI: 10.1039/b413542c
studies.5a Some of these analogues with an extended seven-bond
One-carbon extended conformationally constrained pyrrolidine
backbone cross-pair with RNA with high affinity compared to
DNA.5a,d In contrast, Lowe et al. very effectively accomplished
preferential DNA binding by replacing glycine with one-atom
extended b-amino acids in their prolyl-glycyl PNA analogs.6 We
had earlier reported pyrrolidine based positively charged PNAs
and found that the ring system in either 2R,4R or 2S,4S cis
stereochemistry as a polyamide or chimeric amide-sugar phosphate
backbone destabilized the complexes with either DNA or RNA.4d,e
Our preliminary results on the chimeric phosphate–amide
extended backbone revealed that 2R,4R pyrrolidine–amide
chimera were accommodated better in triplex forming sequences.4f
In view of the above discussion, studies of chiral pyrrolidine PNA
with an extended backbone (Fig. 1) that may have potential for
selective targetting to DNA or RNA was undertaken. The positive
charge and chirality in the backbone could have additional
advantages such as increased water solubility and orientational
selectivity binding.
PNA monomer (bepPNA) has been synthesized, incorporated
into PNA sequences at predefined positions, and showed
selective RNA binding properties.
Among several nucleic acid analogues, Peptide Nucleic Acids
(aegPNA, Fig. 1) have emerged as important DNA/RNA binding
molecules.1 PNAs bind to DNA and RNA with high thermal
stability and specificity by forming duplexes (WC base pair) and
triplexes (WC base pair and HG hydrogen bonding).1 The use of
PNAs as gene-targeted drugs and as tools in molecular biology is a
developing field.2 However, weaknesses of PNAs such as limited
aqueous solubility, poor cellular uptake and binding to cDNA/
RNA in either parallel or antiparallel orientation1b need to be
addressed before they can become viable drug candidates. Our
approach3 so far has been to create chiral, conformationally
restricted PNA backbones that reduce the otherwise possible
conformational states of the flexible open chain single strand
PNAs to those appearing in the complex form. The pyrrolidine
PNA (Fig. 1) is the outcome of one of the ways to lock the PNA
conformation for this purpose. The mixed results surfacing from
different groups4 infer that the conformational constraint imposed
onto the PNA backbone in the form of ring structure may not be
uniform for various nucleobases or with sequence context. The
conformational locking of the backbone probably needs to be
reasonably moderated for fine tuning the internucleobase distance
complementarity for uniform effective recognition via WC/HG
hydrogen bonding. There are many examples in the literature that
suggest that a five-atom amide leading to a seven-atom repeating
backbone may be more useful because of the reduced conforma-
tional flexibility of the amide relative to the six-atom phosphodie-
ster backbone.5 This postulate has also been supported by X-ray
The synthesis of monomer 5 was accomplished as shown in
Scheme 1. Protection of the ring nitrogen of the naturally
occurring trans-4-hydroxy-L-proline and esterification with
MeOH–SOCl2 gave methyl ester 1.4 The methyl ester was reduced
to the diol using LiCl/NaBH4 and selective tosylation of the
primary hydroxy group by controlled dropwise addition of p-TsCl
in pyridine gave monotosylate 2 (ditosylate side product was
removed by column chromatography), which was treated with
excess NaN3 to obtain the azido compound. The selective
hydrogenation of the azide function using Raney-Ni in methanol
gave the free amine and Boc protection of free amine yielded
compound 3. This was then subjected to hydrogenation over Pd–C
to remove the N-benzyloxy carbonyl group and subsequent
alkylation of the free ring nitrogen with ethyl acrylate gave
N-alkylated product 4. The nucleobase thymine was introduced at
C-4 to get the protected monomer ethyl ester by treatment with
Fig. 1 Designed bepPNA as DNA/RNA and PNA mimic.
{ Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis of compounds, 1H, 13C NMR, mass spectral
data, HPLC profiles, pH titration data and melting curves for triplexes.
See http://www.rsc.org/suppdata/cc/b4/b413542c/
Scheme 1 Synthesis of protected bepPNA monomer.
Chem. Commun., 2005, 495–497 | 495
*vakumar@dalton.ncl.res.in
This journal is ß The Royal Society of Chemistry 2005

View Article Online
N3-benzoylthymine under Mitsunobu conditions. The ester
hydrolysis and deprotection of N3 of thymine using 2 M NaOH
in methanol–water yielded the required monomer [(2S,4S)-2-(tert-
butoxycarbonylaminomethyl)-4-(thymin-1-yl)pyrolidin-1-yl] pro-
panoic acid
5 in good yield. All new compounds were
characterized by NMR and mass spectrometry. The pKa of the
ring nitrogen was found to be 7.7 and partial protonation at
physiological conditions may be expected.
The monomer 5 was incorporated into oligothymine PNA
sequences at C-/N- terminals, in the center, alternative positions
and through the entire sequence by solid phase peptide synthesis
on L-lysine derivatized MBHA resin using Boc chemistry.2b The
oligomers were cleaved from the solid support using the TFA–
TFMSA method2b to yield bepPNAs 7–11. For control studies
aegPNA T8 octamer 6 was also synthesized. These were purified
by RP HPLC on a semi-preparative C18 column and characterized
by MALDI-TOF mass spectral analysis.7
Fig. 2 PNA–DNA complexation. Lane 1, BPB; lane 2, (aegPNA
6)2:DNA ; lane 3, ssbepPNA 7; lane 4, ssDNA ; lane 5, (bepPNA 7)2
+
DNA; lane 6, (bepPNA 8)2 + DNA; lane 7, (bepPNA 9)2 :DNA; lane 8,
(bepPNA 10)2 + DNA; lane 9, (bepPNA 11)2 + DNA; lane 10, BPB
(bromophenol blue). DNA 5 CGCAAAAAAAACGC. A. PNA–DNA
complex, B. BPB, C. Single stranded DNA.
The Tm values of homopyrimidine PNAs 6–11, hybridized with
complementary DNA and RNA were obtained from temperature
dependent UV-absorbance data (Table 1). UV and CD Job
plots6,8 suggested the formation of 2:1 bepPNA2:DNA and
bepPNA2:RNA triplexes and hence all the complementation
studies were performed with 2:1 PNA:DNA/RNA stoichiometry.
The C-terminal modified bepPNA 7 binds to DNA with slight
decrease in Tm (DTm 5 21 uC) whereas bepPNA 9 modified at the
N-terminal stabilizes the complex (DTm 5 +2 uC) compared to
control aegPNA 6. Surprisingly, bepPNA 8, modified unit at the
center did not show any complexation with DNA. Alternate and
homooligomeric bepPNAs (10 and 11) did not form complexes
with DNA as shown in UV-melting curves. A linear increase in
absorbance was observed in these cases that corresponded to the
PNA and DNA single strand melting (ESI{). UV-Tm data of
PNA:DNA complexation is supported by the gel shift assay
(Fig. 2). The bepPNA 8 exhibited very weak binding interaction at
lower temperature, though it was not seen during UV-Tm thermal
denaturation (Fig. 2, lane 6). When the complexation studies were
performed with RNA, chimeric PNAs with a single bepPNA unit
were found to bind with approximately the same Tm but slightly
lower than that of control aegPNA 6. bepPNA 10 with alternating
Fig. 3 First derivatives of melting curves of (a) aegPNA 6 and chimeric
aeg-bepPNA, (b) 7, (c) 8, (d) 9, (e) 10 and (f) bepPNA 11 with RNA
(poly rA).
but with reduced strength compared to the alternating aeg-bep
PNA.
These results suggest that bepPNA monomer in chimeric and
homooligomeric PNAs induced binding selectivity for RNA over
DNA. Incorporation of the modified units at the terminals (C-/N-)
seems to exert only a very weak effective preorganized conforma-
tion and allowed binding with DNA as well as RNA. When in the
centre of the sequence, the induced conformation allows recogni-
tion of RNA but that of DNA is suppressed. The high affinity
binding of alternating aeg-bepPNA 10 with RNA suggests that the
alternating aeg-bep units are uniformly spaced such that a
balanced optimum conformation may be reached for recognition
of RNA. The fully modified backbone in 11 binds to RNA but
with reduced strength compared with the alternating sequence 10.
This could be because of overpreorganization of the single strand
as suggested for fully modified LNA9 or high positive charge
concentration of two bep-homooligomers in 2:1 binding mode.
The 2:1 binding stoichiometry for 11:RNA was confirmed by a
UV-Job plot (ESI{). The charge–charge repulsions could therefore
be a possible reason for the observed reduced Tm. The inclusion of
an extra atom in the backbone was easily achieved by using
conjugate addition to ethyl acrylate as compared to cumbersome
methods in the sugar–phosphate backbone.5 The selective RNA
recognition by the chiral, cationic PNA analogue is important
for application perspectives. Further studies on the mixed
aeg-bep units exhibited a very high binding affinity (DTm
5
+ 4.5 uC/mod) (Table 1, Fig. 3). The observed transitions were very
sharp with RNA compared to those with DNA. The sequence 11
comprised of only bepPNA backbone also recognized only RNA
Table 1 UV-Tm values in uC of PNA2:DNA/RNA triplexesa
Entry
Sequence
DNA
RNA
1
2
3
4
5
6
aegPNA 6, H-TTTTTTTT-LysNH2
bepPNA 7, H-TTTTTTTt-LysNH2
bepPNA 8, H-TTTtTTTT-LysNH2
bepPNA 9, H-tTTTTTT-LysNH2
bepPNA 10, H-TtTtTtTt-LysNH2
bepPNA 11, H-tttttttt-LysNH2
51.5
49.0
65.8
59.9
59.2
59.0
84.4
58.9
b
53.0
b
b
a
Tm 5 melting temperature (measured in buffer: 10 mM sodium
phosphate, pH 7.0 with 100 mM NaCl and 0.1 mM EDTA).
Measured from 10 to 90 uC at ramp 0.2 uC min21. UV-absorbance
measured at 260 nm. All values are an average of three independent
experiments and accurate to within ¡0.5 uC. DNA 5 CGCA8CGC,
b
RNA 5 polyrA. T 5 aegPNA and t 5 bepPNA monomers. Did
not form complex with DNA.
496 | Chem. Commun., 2005, 495–497
This journal is ß The Royal Society of Chemistry 2005

View Article Online
3 V. A. Kumar, Eur. J. Org. Chem., 2002, 2021–2032.
purine–pyrimidine sequences and other diastereomeric bepPNAs
are in progress.
4 (a) D. T. Hickman, P. M. King, M. A. Cooper, J. M. Slater and
J. Mickelfield, Chem. Commun., 2000, 2251–2252; (b) T. H. S. Tan,
D. T. Hickman, J. Morral, I. G. Beadham and J. Micklefield, Chem.
Commun., 2004, 516–517; (c) A. Puschl, T. Tedeschi and P. E. Nielsen,
Org. Lett., 2000, 2, 4161–4163; (d) V. Kumar, P. S. Pallan, Meena and
K. N. Ganesh, Org. Lett., 2001, 3, 1269–1272; (e) Meena and
V. A. Kumar, Nucleosides, Nucleotides Nucleic Acids, 2003, 22,
1285–1288; (f) V. A. Kumar and Meena, Nucleosides, Nucleotides
Nucleic Acids, 2003, 22, 1101–1104.
V. A. K. thanks the Department of Science and Technology,
New Delhi for research grants. T. G. thanks CSIR, New Delhi for
a fellowship. We thank Dr K. N. Ganesh for support and
encouragement.
T. Govindaraju and Vaijayanti A. Kumar*
Division of Organic Chemistry (Synthesis), National Chemical
Laboratory, Pune, 411008, India. E-mail: vakumar@dalton.ncl.res.in;
Fax: 91 20 589 3153; Tel: 91 20 589 3153
5 (a) C. J. Wilds, G. Minasov, F. Natt, P. Von Matt, K.-H. Altamann
and M. Egli, Nucleosides, Nucleotides Nucleic Acids, 2001, 20, 991–994;
(b) G. V. Petersen and J. Wengel, Tetrahedron, 1995, 51, 2145–2154; (c)
A. Chur, B. Holst, O. Dahl, P. Valentin-Hansen and E. B. Pedersen,
Nucleic Acids Res., 1993, 21, 5179–5183; (d) A. Lauritsen and J. Wengel,
Chem. Commun., 2002, 530–531.
Notes and references
6 T. Vilaivan and G. Lowe, J. Am. Chem. Soc., 2002, 124, 9326–
9327.
1 (a) M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier,
D. A. Driver, R. H. Berg, S. K. Kim, B. Norden and P. E. Nielsen,
Nature, 1993, 365, 566–568; (b) K. N. Ganesh and P. E. Nielsen, Curr.
Org. Chem., 2000, 4, 1931–1943.
2 (a) D. A. Braasch and D. R. Corey, Biochemistry, 2002, 41, 4503–4510;
(b) P. E. Nielsen and M. Egholm, in Peptide nucleic acids (PNA),
Protocols and Applications, ed. P. E. Nielsen and M. Egholm, Horizon
Scientific, Norfolk, CT, 1999.
7 LC-TOF-MS: aegPNA 6, 2275.90 [M + H+]; bepPNA 7, 2288.0
[M + 2H+]; bepPNA 8, 2288.0 [M + 2H+]; bepPNA 9, 2288.04,
[M + 2H+]; bepPNA 10, 2326.06 [M + 3H+]; bepPNA 11, 2374.43
[M + 4H+].
8 P. Job, Ann. Chim. (Paris), 1928, 9, 113–203.
9 C. J. Leumann, Bioorg. Med. Chem., 2002, 10, 841–854.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 495–497 | 497