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.
Fig. 1 ORTEP diagram of the crystal structure of 3.
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
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,
b307362a/ for crystallographic data in CIF or other electronic format.
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.
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.
Table 1 UV-Tm (°C) of PNA-DNA/RNA hybridsa
Entry
PNA
DNA 13
poly(rA)
4 L. Betts, J. A. Josey, J. M. Veal and S. R. Jordan, Science, 1995, 270,
1838.
1
2
3
5
6
8
9
10
11
12
34.8 (14)
58.0 (39)
43.1 (19)
41.8 (14)
45.6 (8)
35.1
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.
50.7 (12)
50.9 (12)
53.3 (10)
> 80
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.
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