New PNA analogues derived from aminoethylpyrrolidin- 5-one backbone show stabilization of aepone-PNA:DNA hybrids and destabilization of the corresponding RNA hybrids compared to unmodified PNA

Ganesh, Krishna N.

Sharma, Nagendra K.

English

Publications of the IAS Fellows

Expanding the repertoire of pyrrolidyl PNA analogues for DNA/RNA
hybridization selectivity: aminoethylpyrrolidinone PNA (aepone-PNA)†
Nagendra K. Sharma and Krishna N. Ganesh*
Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, India.
E-mail: kng@ems.ncl.res.in; Fax: 91 20 589 3153; Tel: 91 20 589 3153
Received (in Cambridge, UK) 30th June 2003, Accepted 7th August 2003
First published as an Advance Article on the web 20th August 2003
New PNA analogues derived from aminoethylpyrrolidin-
5-one backbone show stabilization of aepone-PNA:DNA
hybrids and destabilization of the corresponding RNA
hybrids compared to unmodified PNA.
Peptide Nucleic Acids (PNA, I) are one of the most prominent
findings of the search for structural analogues of DNA/RNA for
antigene/antisense therapeutics.1 The binding of PNA to the
target DNA/RNA sequences occurs with high sequence speci-
ficity and this attribute of PNAs for biological and medicinal
applications has not yet been fully realized due to poor aqueous
solubility, ambiguity in binding orientation specificity (parallel/
antiparallel) and lack of sufficient discrimination in binding
between target DNA and RNA.1b,2 Classical PNA is conforma-
tionally very flexible and can attain different conformations to
accommodate binding to both DNA and RNA. NMR study of
PNA oligomers has indicated it to be a complex mixture (up to
2n) of conformational isomers arising from cis and trans tertiary
amide bonds with a significant barrier to rotational inter-
conversion.3 PNA hybridization to DNA/RNA is dependent on
the tertiary amide conformation and hence affected by the slow
rotamer equilibrium. Examination of the crystal structures of
PNA/DNA, PNA/RNA, PNA/PNA and PNA2/DNA triplexes
reveal that the linker carbonyl is pointing towards the carboxyl
end of PNA.4
One way to study the criticality of such structural features is
to design structures with frozen rotation of the side chain by
locking them into rings as exemplified by the different
pyrrolidene based PNA analogues.5 In one of our earlier
modifications, remarkable stabilization of the derived
PNA:DNA hybrids was achieved in the chiral and cationic
aminoethylprolyl PNA (aep-PNA, II), having the neutral
tertiary amide group of PNA I replaced by a protonatable cyclic
tertiary amine.6 In order to avoid the dominance of the non-
sequence specific electrostatic component in aep-PNA:DNA
binding and to get the best characteristics from both the normal
PNA and the aep-PNA, we resorted to restoring the amide
character to the pyrrolidene ring nitrogen. Herein we report the
synthesis and evaluation of aminoethylpyrrolidin-5-one PNA
(aepone-PNA, III) having the endocyclic amide CO at C5. The
synthesis of all four nucleobase protected monomers (4,5) and
incorporation of the aepone-T monomer 4 into aeg-PNA
backbone to examine the selectivity in hybridization stability
with DNA and RNA is reported. Our present modification with
a pyrrolidine-N1–CH2CH2NH– backbone is quite different
from the previously known7 pyrrolidine-N1–CH2CO backbone
(IV) as it leads to different C and N termini for the derived
PNAs. The chemical synthesis of the target pyrrolidin-5-one
system was done via selective C5-oxidation of N-(aminoethyl-
)prolines, wherein despite the available choice of endo- and
exo-cyclic Na–CH2 groups, the endocyclic 5-CH2 is preferen-
tially oxidized as confirmed by the reported crystal structure.
The literature methods for the synthesis of 4-substituted
pyrrolidin-5-one consist of reaction of proline substrates with
RuO4 generated in situ by oxidation of RuO2 with NaIO4.8 In
these examples, the ring imino nitrogen is protected with a Boc
group. In our examples, N1 has an ethylamino substituent that
has competing Na-methylene groups susceptible to oxidation.
The reaction of (4R)-O-mesyl-N1(ethylamino-N-boc) proline
ester 2 with RuCl3/NaIO4, in a biphasic solvent system, was
complete within 1 h and led to a product mixture from which
only the major C5-one product 3 could be isolated in 45% yield
and successfully characterized (Scheme 1). The identity of the
oxidation site in 3 as C5 was unambiguously deduced from its
crystal structure (Fig. 1).‡ The oxidation of the C5 endocyclic
methylene seems to be preferred over that of exocyclic
methylenes, in spite of C5 having an electronegative a-
substituent. This compound was used to alkylate N1 of
pyrimidines T and C and N9 of purines A and 2-amino-
6-chloropurine (precursor for G) which are suitably protected at
exocyclic amino groups (Scheme 1) to obtain the (2S,4S)-
aepone-PNA monomer esters (4a–7a). Upon hydrolysis with
LiOH/MeOH esters yielded the monomers (4b–7b) suitable for
solid phase synthesis of aepone-PNA oligomers. It should be
pointed out that the synthetic strategy for pyrrolidinone PNA
monomers reported here involving prior N-alkylation followed
by C5 oxidation is much shorter than the one previously
reported7 for similar analogues.
† Electronic supplementary information (ESI) available: 1H NMR of 3–7,
mass spectra of 3–7, 9–11, HPLC, UV-melting curves and experimental
details. See http://www.rsc.org/suppdata/cc/b3/b307362a/
Scheme 1 Synthesis of aepone-PNA monomers. a) MeSO2Cl, Et3N in DCM
at 0 °C; b) NaIO4, RuCl3·xH2O, CH3CN–CCl4–H2O (1 : 1 : 1.5), 20 min; c)
K2CO3, 18-crown-6 ether, DMF, 70 °C; i) thymine; ii) N4-cbz-cytosine; iii)
N6-bz-adenine; iv) 2-amino-6-chloropurine.
Th is journa l i s © The Roya l Soc ie ty of Chemist ry 20032484 CHEM. COMMUN. , 2003, 2484–2485
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PNA T8 oligomers 9–12 incorporating the modified mono-
mers were synthesized using Boc chemistry on b-alanine
derivatized Merrifield resin followed by cleavage from the resin
with TFA/TFMSA, purification of PNA oligomers by reverse
phase HPLC and characterized by MALDI-TOF. The modified
aepone-T monomer 4b was incorporated at the C-terminus in
PNA 9, at the C-terminus and centre in PNA 10 and at all
positions in PNA 11. The complementary DNA sequence 13
(GCA8CG) had GC and CG locks at the 5A- and 3A-ends to avoid
slippage of duplexes. The PNA:DNA/RNA complexes were
constituted by mixing appropriate strands in a 2 : 1 stoichio-
metry in buffer followed by heating to 90 °C and annealed by
slow cooling to 4 °C to obtain PNA2:DNA triplexes.
The Tms of different triplexes as extracted from the derivative
plot of temperature dependent UV absorbance (Fig. 2) at 260
nm is shown in Table 1. It is seen that aepone-PNA oligomers
9–11 significantly stabilise the derived triplexes with DNA 14
as compared to that from the unmodified PNA oligomer 8 (DTm
16–19 °C) (Fig. 2A). In comparison, the aepone-PNAs 9–11
effected destabilization of the triplexes formed with poly(rA),
compared to the triplex from unmodified PNA 8 (DTm 12–15
°C) (Fig. 2B). What is significant is that even the completely
modified PNA oligomer 11 binds DNA and poly(rA) with a
well defined Tm. This result on specificity of hybridization of
aepone-PNAs 9–11 with preference for significant stabilization
of DNA hybrids over RNA hybrids of unmodified PNA 8 is
opposite to the selectivity observed for pyrrolidinone-A8 PNA
with opposite polarity;7 these analogues stabilised RNA hybrids
more than the DNA hybrids. The aepone-PNA analogues are
more akin to the recently reported9 pyrrolidinyl PNAs in terms
of observed selectivities.
8. H2N–T–T–T–T–T–T–T–T-b-ala-COOH (aeg-T8)
9. H2N–T–T–T–T–T–T–T-t-b-ala-COOH
10. H2N–T–T–T-t-T–T–T-t-b-ala-COOH
11. H2N-t-t-t-t-t-t-t-t-b-ala-COOH (aepone-t8)
12 H2N-t-t-t-t-t-t-t-t-b-ala-COOH (aep-t8)
13 d(GCAAAAAAAACG) (DNA)
The aep-PNA oligomer 12 devoid of C5 carbonyl, bound
DNA with a very high Tm, melting incompletely even at 80 °C.
The strong binding of 12 with DNA is not entirely due to the
electrostatic interactions as it showed a lower binding with
poly(rA) as compared to PNA 8. This suggests that the
conformational preorganization plays an important role in
determining the binding strengths. In this context, the binding
pattern of the presently designed aepone-PNA is interesting; it
has affinity to DNA more than that of PNA, but lower than that
of aep-PNA and affinity to RNA less than that of PNA and more
than that of aep-PNA. The tetrahedral nature of pyrrolidine
nitrogen in aep-PNA is switched back to the planar amide in
aepone-PNA, as in unmodified PNA with a consequent
influence on the backbone conformation. Importantly, the side-
chain syn/anti rotameric equilibrium present in unmodified
PNA is not possible in aepone-PNA, although the ring nitrogen
retains the amide character. Thus aepone-PNA (III) is an
evolved structure by design, combining the features of both
PNA (I) and aep-PNA (II). It also emerges from the present
data that aep-PNA has a selectivity to bind DNA over RNA, and
this aspect needs to be confirmed with studies using mixed RNA
sequences. The CD spectral features of aepone-PNA:DNA/
RNA hybrids were similar to that of PNA:DNA/RNA hybrids,
suggesting no major differences in base stacking patterns.
In summary, we have reported the synthesis of (2S,4S)-
aepone-PNA monomers (4–7) as new PNA analogues via
selective C5 oxidation of aep-proline derivatised intermediate
2. The aepone-poly T8 oligomers (9–11) show reverse selectiv-
ity in DNA/RNA binding compared with the reported glycyla-
minomethyl pyrrolidinone7 and are a useful addition to the
growing library of proline/pyrrolidine based PNA analogues5 to
fine tune the binding selectivities. Further studies to delineate
the sequence dependent effects of aepone-PNA and its
stereomers are in progress.
N. K. S. thanks UGC, New Delhi for a research fellowship.
We thank Dr M. Bhadbhade and Mr R. Gonnade for crystal
data.
Notes and references
‡ Crystal data for 3: Crystallised from CH2Cl2–MeOH, C14H24N2O8S, M =
380.41, crystal dimensions 0.61 3 0.09 3 0.05 mm, crystal system:
monoclinic, space group P21, a = 12.739(5), b = 9.294(4), c = 15.994(6)
Å, b = 103.419(8)°, V = 1841.9(13) Å3, Z = 4, Dc = 1.372 g cm23, m(Mo-
Ka) = 0.219 mm21, T = 293(2) K, F(000) = 808, max. and min.
transmission 0.9885 and 0.8780, 9094 reflections collected, 6134 unique [I
> 2s(I)], S = 1.109, R value 0.0652, wR2 = 0.1213 (all data R = 0.0816,
wR2 = 0.1283). CCDC 213533. See http://www.rsc.org/suppdata/cc/b3/
b307362a/ for crystallographic data in CIF or other electronic format.
1 (a) P. E. Nielsen and G. Haaima, Chem. Soc. Rev., 1997, 73 and
references cited therein (b) P. E. Nielsen and K. N. Ganesh, Curr. Org.
Chem., 2000, 4, 931.
2 D. A. Braasch and D. R. Corey, Biochemistry, 2002, 41, 4503.
3 S. C. Brown, S. A. Thomson, J. M. Veal and D. G. Davis, Science, 1994,
265, 777.
4 L. Betts, J. A. Josey, J. M. Veal and S. R. Jordan, Science, 1995, 270,
1838.
5 V. A. Kumar, Eur. J. Org. Chem., 2002, 2021–2032.
6 (a) M. D’Costa, V. A. Kumar and K. N. Ganesh, Org. Lett., 1999, 1,
1513; (b) M. D’Costa, V. A. Kumar and K. N. Ganesh, Org. Lett., 2001,
3, 1281.
7 A. Puschl, T. Boesen, G. Zuccarello, O. Dahl, S. Pitsch and P. E. Nielsen,
J. Org. Chem., 2001, 66, 707.
8 (a) X. Zhang, A. C. Schmitt and W. Jiang, Tetrahedron Lett., 2001, 42,
5335; (b) X.-L. Qiu and F.-L. Qing, J. Org. Chem., 2003, 68, 3614.
9 T. Vilaivan and G. Lowe, J. Am. Chem. Soc., 2002, 124, 9326.
Fig. 1 ORTEP diagram of the crystal structure of 3.
Fig. 2 Derivative UV absorbance (260 nm)–temperature profiles. A)
PNA:DNA13 hybrids and B) PNA:poly(rA) hybrids: a) 8, b) 9, c) 10, d)
11.
Table 1 UV-Tm (°C) of PNA-DNA/RNA hybridsa
Entry PNA DNA 13 poly(rA)
1 8 34.8 (14) 58.0 (39)
2 9 50.7 (12) 43.1 (19)
3 10 50.9 (12) 41.8 (14)
5 11 53.3 (10) 45.6 (8)
6 12 > 80 35.1
a Buffer: 10 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA. The
values quoted are the average of three experiments and are accurate to ±0.5
°C. Values in parentheses indicate %hyperchromicities.
2485CHEM. COMMUN. , 2003, 2484–2485


Expanding the repertoire of pyrrolidyl PNA analogues for DNA/RNA hybridization selectivity: aminoethylpyrrolidinone PNA (aepone-PNA)

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Expanding the repertoire of pyrrolidyl PNA analogues for DNA/RNA hybridization selectivity: aminoethylpyrrolidinone PNA (aepone-PNA)

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