Gokhale et al.
JOCNote
TABLE 2. UV Tm (°C) Data for TANA and iso-TANA Modified DNA
thioacetamido linker are more compatible for RNA binding
when in ribo configuration and (2) the different linker folding
geometry in the xylo configured amino sugar in the 5-atom
thioacetamido linked dimers (iso-TANA) seems to be more
favorable for DNA binding over that for RNA. The backbone
chirality in thioacetamido linker thus is able to sense the subtle
structural differences in DNA and RNA. The RNA selectivity of
binding of TANA-modified oligomers could be reversed to
DNA binding selectivity by the change in configuration at the
30-amino-substituted sugar. The significance of the thioacetami-
do linker in either ribo- or xylo-sugar configurations is thus
evident, and is in contrast to the 3-xylo-configured phosphodie-
ster linked oligonucleotides, where no co-operative transition
was observed in the mixed purine-pyrimidine duplexes with
either RNA or DNA.
Sequences with Complementary DNA and RNAa
UV Tm (°C) for iso-TANA
UV Tm (°C) for TANA
DNA-2
RNA-2
DNA-2
RNA-2
DNA-1
DNA-3
DNA-4
54.6
49.3
42.6
53.8
46.7
40.9
54.7
39.84a
43.54a
54.7
47.54a
52.84a
UV Tm (°C) for iso-TANA
UV Tm (°C) for TANA
DNA-6
RNA-6
DNA-6
RNA-6
DNA-5
DNA-7
DNA-8
55.3
43.3
44.2
55.5
38.7
33.2
54.6
43.2
41.6
55.1
54.5
43.6
aThe complexes were prepared in 10 mM sodium phosphate buffer,
pH 7.2 containing NaCl (100 mM) and EDTA (0.1 mM). All values are
an average of at least three experiments and accurate to within (1.0°C.
DNA-2:50 TGTAACTGAGGTAAGAGG 30, RNA-2:50 UGU AACU-
GAGGUAAGAGG 30, DNA-6:50 AAGAGTTCAAAAGCC CTTC
30, RNA-6:50 AAGAGUUCAAAAGCCCUUC 30.
Experimental Section
Synthesis of iso-TANA Dimers. S-50-(30-O-TBS-thymidinyl)-
mercapto acetic acid 7(1 g, 2.32 mmol), TBTU (0.89 g, 2.784 mmol),
and HOBt (0.15 g 1.16 mmol) were taken in dry CH3CN:dry DMF
(5:1) (5 mL), diisopropylethyl amine (1.00 mL, 5.8 mmol) was
added, and the solution was stirred for 15 min. Then corresponding
amino compound 2a/6a (2.32 mmol) dissolved in 4 mL of CH3CN
was added into the reaction mixture with a further 2 h of stirring.
Then the reaction mixture was concentrated and the product was
extracted by the usual workup procedure. 30-O-TBS protection of
dimers (8a/9a) was removed with 1 N TBAF in THF and the prod-
uct was purified by column chromatography. These dimers were
then converted into their phosphoramidite derivatives (8c/9c). The
protecting groups in the dimers (8a/9a) were removed appropri-
ately to give dimers (8b/9b). All the nucleoside sugar protons were
assigned by 2D COSY experiments These dimers were used for
sugar conformational analysis by NMR and for CD analysis.
NMR and HRMS Analysis for Dimer 8b. 1H NMR (200 MHz,
D2O) δ 1.75 (s, 6H, TA, TB, CH3), 2.04-2.08 (m, 1H, TA, H20),
2.21-2.31 (m, 2H, TB, H20, H20), 2.66-2.73 (m, 2H, TA, H200, TB,
H50), 2.86-2.89 (m, 1H, TB, H500), 3.20-3.28 (dd, 2H, SCH2CO,
J=15.65 Hz), 3.63-3.75 (m, 2H, TA, H50, H500), 3.86-3.90 (m,
1H, TB, H40), 4.08-4.11 (m, 1H, TA, H40), 4.23-4.27 (m, 1H, TB,
H30), 4.49-4.53 (m, 1H, TA, H30), 5.86-5.89 (m, 1H, TA, H10, J =
5.87 Hz, 7.34 Hz), 6.06-6.09 (m, 1H, TB, H10, J = 6.85 Hz, 6.84
Hz), 7.35, 7.59 (s, 2H, TA, TB, H6). Mass calcd. 562.15, obsd 562.21.
HRMS calcd for (C22H29N5O9NaS) 562.1583, obsd 562.1572
NMR and HRMS Analysis for Dimer 9b. 1H NMR (200 MHz,
D2O) δ δ 1.84, 1.92 (s, 6H, CA, TB, CH3), 2.10-2.14 (m, 1H, CA,
H20), 2.31-2.40 (m, 2H, TB, H20,H20), 2.77-2.83 (m, 2H, CA,H200,
TB H50), 2.91-2.95 (m, 1H, TB, H500), 3.31-3.38 (m, 2H,
SCH2CO), 3.75-3.88 (m, 2H, CA, H50, H500), 3.95-3.99 (m, 1H,
TB, H40), 4.19-4.22 (m, 1H, CA, H40), 4.23-4.35 (m, 1H, TB, H30),
4.56-4.60 (m, 1H, CA, H30), 5.91-5.94 (m, 1H, CA, H10, J = 5.8
Hz, 6.8 Hz), 6.14-6.18 (m, 1H, TB, H10, J = 6.7 Hz, 6.7 Hz), 7.44,
7.68 (s, 2H, CA, TB, H6). Mass calcd 561.17, obsd 561.20, 585.57.
HRMS calcd for (C22H30N6O8NaS) 561.1743, obsd 562.1747,
calcd for (C22H30N6O8KS) 577.1692, obsd 577.1679
These oligonucleotides containing either TANA or iso-TANA
dimer units (DNA-3/4 and DNA-7/8) were subjected to thermal
denaturation-UV measurement experiments for testing their
binding affinity to complementary sequences DNA-2/RNA-2 or
DNA-6/RNA-6, respectively, and the results are summarized in
Table 2. The unmodified DNA-1 and DNA-5 bind to comple-
mentary (DNA-2 and RNA-2) and (DNA-6 and RNA-6),
respectively, with almost equal strength as noted by the UV-Tm
values. Incorporation of one (DNA-3) or two (DNA-4) TANA
dimer units in DNA-1 seems to stabilize the duplexes with RNA-2
more effectively as compared to their duplexes with complemen-
tary DNA-2. In another DNA sequence, DNA-5,three(DNA-7),
or four (DNA-8) modified TANA units were incorporated. The
duplex of DNA-7 with RNA-6 was found to be almost as stable
as unmodified duplex DNA-5:RNA-6 but the complex of DNA-7
with DNA-6 was much more destabilized (ΔTm=-11.3 °C).
These result are similar to the other TANA-DNA chimera
that we reported earlier.4 Unexpectedly, the modified
sequence with four modified TANA units, DNA-8, in which
the second consecutive TANA CsT unit was incorporated in
addition to the three TANA blocks in DNA-7, formed the
duplex with complementary RNA-6 with lower thermal stability
(ΔTm=-10.9 °C). In contrast, with incorporation of iso-TsT
and iso-CsT dimer blocks in the same sequences, i.e., in DNA-1
(DNA-3, DNA-4) and in DNA-5 (DNA-7, DNA-8), the com-
plexes with cDNA (DNA-3/4:DNA-2 or DNA-7/8:DNA-6) were
found to be more stable compared to complexes with com-
plementary RNA (DNA-3/4:RNA-2, DNA-7/8:RNA-6) with
ΔTm=-2 to 4 °C and -5 to 11 °C, respectively.
Our results from NMR studies point out the shift toward
S-type sugar conformations in 5-atom thioacetamido linked
dimer blocks in either ribo- or xylo (iso)-configured amino
sugars. CD studies point out the differences in the geometries
of the stacked forms in the two ribo/xylo (iso)-configured
dimers, which could arise from differences in the folding geom-
etry of the amide linker in two isomeric forms. Incorporation
of these TANA/iso-TANA dimers in the mixed purine-pyrimi-
dine oligomers allows them to differentiate between DNA and
RNA backbones, TANA modifications favoring RNA binding
and iso-TANA favoring DNA binding. Thus, on the basis of
temperature-dependent CD and UV melting experiments, it
could be concluded, in the context of the sequences studied,
that (1) the internucleoside distances due to the extended 5-atom
Acknowledgment. S.S.G. thanks UGC, New Delhi for a
senior research fellowship. V.A.K. thanks CSIR, New Delhi
for a research grant (NWP0036).
Supporting Information Available: Synthetic procedures,
1H, 13C, and mass spectra of selected compounds in Scheme 1,
HRMS and 2D COSY spectra of compounds 8b and 9b, HPLC
and MALDI-TOF analysis of modified sequences, and UV-Tm
graphs for the derived complex. This material is available free of
7434 J. Org. Chem. Vol. 75, No. 21, 2010