Sugar-thioacetamide backbone in oligodeoxyribonucleosides for specific recognition of nucleic acids

Article abstract of DOI:10.1039/b603958h

The amide linkage being shorter than the natural phosphate linkage, an additional atom is introduced into oligodeoxyribonucleosides (ODNs) with sugar-thioacetamide backbone that show very good RNA recognition properties. The Royal Society of Chemistry 200

Full text of DOI:10.1039/b603958h

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COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Sugar–thioacetamide backbone in oligodeoxyribonucleosides for
specific recognition of nucleic acids{
Khirud Gogoi, Anita D. Gunjal and Vaijayanti A. Kumar*
Received (in Cambridge, UK) 16th March 2006, Accepted 4th April 2006
First published as an Advance Article on the web 25th April 2006
DOI: 10.1039/b603958h
The amide linkage being shorter than the natural phosphate
linkage, an additional atom is introduced into oligodeoxyr-
ibonucleosides (ODNs) with sugar–thioacetamide backbone
that show very good RNA recognition properties.
bepPNA) suggested that ODNs containing an extra atom in the
pyrrolidine–amide backbone stabilized the complexes with RNA
1
0
over DNA. In the search for an ideal backbone, we comply that
seven-atom extended sugar–amide backbones could be more
uniform with respect to nucleobase orientation as well as distance
complementarity to bind complementary RNA sequences. We
employ a very easy synthetic methodology that fulfills the above
mentioned criteria and is much simpler to execute compared to
The use of modified nucleic acids as gene-targeted drugs and as
1
tools in molecular biology is a developing field. The designed
oligodeoxyribonucleosides (ODNs) to be effective leads as drugs
need to have enhanced strength of hybridization with target RNA,
aqueous solubility, efficiency of cellular uptake, degradation of
RNA by RNAs-H enzyme and also easy synthetic methodologies.
The current phosphorothioate ODN drugs are a mixture of
diastereomeric ODNs due to the chirality at the phosphorus. The
design and synthesis should also fulfil the requirement for
enantiomeric purity to obtain homogeneous ODNs. The backbone
modifications being developed presently include the second and
1,11
several other reported oligonucleotide analogues so far.
In this
communication we present the synthesis of thioacetamido nucleic
acids (TANA) and the thermal stability studies with complemen-
tary DNA and RNA sequences. The strategy of the design,
synthesis of the monomer blocks, oligomer synthesis and their
m
complementary RNA recognition using UV-T measurements is
discussed.
The design is based on the following considerations: (1) in
2
third generation antisense ODNs such as 29-O-alkyl, 29-
3
9-deoxy-39amino ribose sugar the five-membered heterocyclic ring
3
O-methylthioethyl, LNA, HeNA, morpholino NA, ami-
4
5
6
12
pucker is preferred to be 39-endo which is better suited for
7
noethylglycyl PNA (Fig. 1, aegPNA) or several PNA modifica-
mRNA recognition; (2) the solid phase synthesis and scale-up
methodology for an amide bond formation are very well
established; (3) the linker group has no chiral center; and (4) the
flexibility of the phosphate group in DNA/RNA may be conserved
by using a five-atom amide linker.
8
tions. The polyamide backbone ODNs such as aegPNA are
promising because of the ease of their synthesis and strong binding
to the target complementary nucleic acids. Among the sugar–
amide backbones, there are many examples in the literature
suggesting that a five-atom amide linker leading to a seven-atom
repeating backbone may be more useful because of the reduced
conformational flexibility of the amide relative to the six-atom
The monomer synthons 39-Fmoc-amino-59-thioacetic acid
derivative of thymidine 6 and 39-Fmoc-amino-59-thioacetic acid
derivative of 29-deoxy cytidine 14 were synthesized from AZT as
shown in Scheme 1 and Scheme 2, respectively. The AZT, 1 was
converted to its 59-tosyl derivative 2. The 39-azido-59-tosyl
thymidine 2 was converted to 59-ethyl thioacetate 3 by nucleophilic
displacement with ethyl thioacetate in the presence of sodium
hydride in very good yields. The azide group was converted to a
9
phosphodiester backbone. This postulate has also been supported
9
by X-ray studies. Some of the analogues with extended seven-
atom backbones cross-pair with RNA with high affinity compared
9
to DNA. Our recent work on PNA modifications (Fig. 1,
Fig. 1 Designed TANA as a DNA/RNA mimic.
Division of Organic Chemistry (Synthesis), National Chemical
Laboratory, Pune, 411008, India. E-mail: va.kumar@ncl.res.in;
Fax: +91 20 25602623; Tel: +91 20 25602623
1
13
Scheme 1 Synthesis of the thyminyl–sugar–amino acid monomer unit.
{
Electronic supplementary information (ESI) available: H, C NMR,
Reagents: (a) tosyl chloride–pyridine; (b) ethyl thioacetate, NaH, DMF;
mass spectral data for compounds 6 and 14, HPLC profiles and mass
spectra for the modified oligomers 15–20, and Job’s plot and melting
curves for the complexes with RNA. See DOI: 10.1039/b603958h
(
c) H
2
S–pyridine–15%TEA; (d) aqueous LiOH (2 M), MeOH; and (e)
, acetone–water.
Fmoc–succinimide, NaHCO
3
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Chem. Commun., 2006, 2373–2375 | 2373

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Table 1 The TANA and TANA–PNA oligomer sequences
a
b
Sequence
RP-HPLC
(Masscalc)/(Massobs)
+
H-t t t t t t t t-bala 15
H-t t t t c t t t-bala 16
H-t t t t t t t t-bala 17
H-t t t t t t t t-bala 18
H-t t t t t t t t-bala 19
H-t t t t t t t t-bala 20
a
12.7
12.3
10.0
8.3
10.1
7.6
2467.6/2491.0 (M + Na )
+
2466.1/2487.9 (M + Na )
+
2249.4/2251.7 (M + H )
+
2249.4/2252.6 (M + H )
+
2342.9/2367.9 (M + Na )
+
2218.2/2218.9 (M + H )
t and c denote TANA backbone, t denotes aegPNA backbone.
b
Retention time in minutes.
3
was further treated with concentrated NH to deprotect the
4
C -amino protecting group of the cytosine residue. The uncharged
backbone polypyrimidine PNA sequences are known to bind to
1
5
complementary DNA/RNA in a triplex mode. To establish the
binding stoichiometry of the TANA oligomers (15 and 16) Job’s
16
plot study was undertaken with complementary DNA/RNA
Scheme 2 Synthesis of the 5-methylcytosinyl–sugar–amino acid mono-
mer unit. Reagents: (a) TBDMSCl, imidazole, DMF; (b) 1,2,4 triazole,
sequences.{ The binding stoichiometry could be clearly established
to be 2 : 1 between sequence 15 and RNA 22 using UV
spectroscopy.{ The TANA : DNA binding could not be observed
in the Job’s plot. All the binding studies of the sequences listed in
Table 1 with complementary DNA/RNA were carried out in 2 : 1
stoichiometry using UV-Tm experiments. The complexes of
TANA 15 with DNA (39-GCAAAAAAAACG-59) 21 and
RNA r(39-GCAAAAAAAACG-59) 22 were used to study their
comparative binding efficiency. The TANA 16 has a 5-methyl
cytosine unit and its binding with complementary DNA
POCl
TBAF, THF; (f) i. tosyl chloride–pyridine and ii. ethyl thioacetate, NaH,
DMF; (g) H S–pyridine–15%TEA; (h) aqueous LiOH (2 M), MeOH; (i)
Fmoc–succinimide, NaHCO , acetone–water.
3 3
, TEA, acetonitrile; (c) conc. NH , dioxane; (d) BzCl, pyridine; (e)
2
3
1
3
3
2
9-amino group in 4 using pyridine–H S in 75% yield. The ester
function was then hydrolyzed to get the free sugar–amino acid 5.
The free amine group was then protected as Fmoc to get 6.
The 5-methyl cytosine monomer 14 was synthesized starting
from AZT (Scheme 2). AZT 1 was protected as 59-O-tert-
butyldimethylsilyl ether to get 7. The nucleobase was then
(39-GCAAAAGAAACG-59)
and
RNA
sequence
r(39-GCAAAAGAAACG-59) 23 was studied. For single nucleo-
tide mismatch studies the UV-Tm measurements were carried out
using sequences 15 : 23 and 16 : 22 (see Supplementary
information,{ normalized UV absorbance vs. temperature plots).
The results are summarized in Table 2. For control, the Tm of
complexes of DNA 39-TTTTTTTT-59 24 and aegPNA 20 with
DNA 21 and RNA 22 were measured under identical buffer
conditions. It is evident from the above experiments that the
homopyrimidine TANA backbone oligomers (15 and 16) bind to
the homopurine complementary RNA sequences (22 and 23,
respectively) and show a single base-mismatch discrimination of
about 10–14 uC. The thermal stability of 16 : 23 was increased by
4
transformed to 5-methyl cytosine derivative 9 via C -1,2,4 triazolo
derivative 8 followed by replacement of the triazolide with
1
4
ammonia to get 9. The exocyclic amino group was benzoyl
protected to get 10. 59-O-desilylation, tosylation and conversion to
59-thioethylacetate derivative of cytosine 11 was accomplished as in
the case of synthesis of thyminyl monomer. The azide group was
converted to 39-amino group using pyridine–H S in 75% yield to
2
1
3
get 12. The ester function was then hydrolyzed to get free sugar–
amino acid 13. It was found that during the basic hydrolysis of the
ester group the exocyclic amino group was deprotected. Protection
of the 39-amino group was then accomplished to get Fmoc–amino
acid 5-methyl cytosine derivative 14. All the new compounds were
1
13
C
characterized using spectroscopic techniques such as IR, H,
a
Table 2 UV-T
RNA triplexes
m
values in uC of (TANA)
2
/(TANA-PNA)
2
: DNA/
NMR and MALDI-TOF mass spectral analysis.{ The protected
thymine monomer 6 and cytosine monomer 14 were utilized in the
solid phase oligomer synthesis.
b
No Sequence
DNA 21 RNA 22
RNA 23
To test the sequence specific DNA/RNA recognition by the
oligomers comprising the sugar–amino acids 6 and 14, two 8-mer
sequences 15 and 16 were synthesized using rink amide resin and
Fmoc peptide synthesis strategy. During the synthesis of sequence
d
c
1
2
3
4
5
6
7
a
H-t t t t t t t t-bala 15 nde
H-t t t t c t t t-bala 16 nd
H-t t t t t t t-bala 17
63.8 (63.5)
52.9
53.5
32.4
25.5
49.8
63.1 (67.1)
—
—
—
52.5
nd
c
nd
H-t t t t t t t t-bala 18 nd
H-t t t t t t t t-bala 19 nd
16, excess of 5-methylcytosine monomer was used in the coupling
H-t t t t t t t-bala 20
39-TTTTTTTT-59 24
42.6
nd
61.5
20
stage and capping with acetic anhydride was done in order to
4
ensure that the free C -amino group remains protected during
Tm = 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 min . UV-absorbance
measured at 260 nm. All values are an average of 3 independent
further synthesis. In order to test the compatibility of these
modified monomers in aegPNA, three sequences with mixed
TANA–PNA backbone (17–19) and control aegPNA 20 were
synthesized using Fmoc based solid phase peptide synthesis
procedures. The oligomer sequences (Table 1) were cleaved from
the solid support using standard conditions and the sequence 16
2
1
b
experiments and accurate to within ¡0.5 uC. T denotes DNA
backbone. Values in parentheses denote melting temperature at pH
c
d
.5. nd = not detected. With complementary DNA sequence
e
5
(
39-GCAAAAGAAACG-59).
2
374 | Chem. Commun., 2006, 2373–2375
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+
15
uC at pH 5.5 in which a C GC base triple is formed. The
4
Notes and references
complex of TANA oligomer 15 with cRNA 22 was highly stable
compared to the DNA : RNA complex (24 : 22) and the stability
1
D. A. Braasch and D. R. Corey, Biochemistry, 2002, 41, 4503–4510;
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; S. T. Crooke, in Antisense Research and
Application, ed. S. T. Crooke, Springer, Berlin, 1998, vol. 131, pp. 1–50;
J. Kurreck, Eur. J. Biochem., 2003, 270, 1628–1644.
2
was comparable with that of aegPNA : RNA (20 : 22). The
formation of cDNA : TANA complexes was not observed. To test
the compatibility of the TANA backbone in aegPNA : TANA
mix-backbone oligomers, the sequences 17–19 were synthesized.
Sequence 17 has a TANA monomer unit at C-terminus, sequence
2 M. Manoharan, Biochim. Biophys. Acta, 1999, 1489, 117–130.
3
T. P. Prakash, M. Manoharan, A. M. Kawasaki, A. S. Fraser,
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Biochemistry, 2002, 41, 11642–11648.
1
8 has the modified unit at the central position and sequence 19 is
an alternate aeg-TANA sequence. All the complexes of oligomers
7–19 with RNA 22 were destabilized compared to the control
aegPNA : RNA complex 20 : 22. Contrary to the earlier reported
4 M. Petersen and J. Wengel, Trends Biotechnol., 2003, 21, 74–81.
5 E. Lescrinier, R. Esnouf, J. Schraml, R. Busson, H. A. Heus,
C. W. Hilbers and P. Herdewijn, Chem. Biol., 2000, 7, 719–731.
6
7
1
J. Summerton, Biochim. Biophys. Acta, 1999, 1489, 141–158.
P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science, 1991,
254, 1497–1500; P. E. Nielsen, M. Egholm and O. Buchardt,
Bioconjugate Chem., 1994, 5, 3–7.
V. A. Kumar, Eur. J. Org. Chem., 2002, 2021–2032; V. A. Kumar and
K. N. Ganesh, Acc. Chem. Res., 2005, 38, 404–412.
J. Wilds, G. Minasov, F. Natt, P. Von Matt, K.-H. Altamann and
M. Egli, Nucleosides Nucleotides Nucleic Acids, 2001, 20, 991–994;
G. V. Petersen and J. Wengel, Tetrahedron, 1995, 51, 2145–2154;
A. Lauritsen and J. Wengel, Chem. Commun., 2002, 530–531; T. Vilaivan
and G. Lowe, J. Am. Chem. Soc., 2002, 124, 9326–9327.
11
bepPNA backbone, the mixed backbone containing sugar amide
and aegPNA seems to be incompatible to form regular helical
structures and mixed backbone ODNs 17–19 show significant
decrease in binding with cRNA. The stability of the complexes of
homogeneous backbone TANA with RNA over DNA is a very
valuable result from an application perspective. The synthesis of
mixed purine–pyrimidine backbone sequences and the study of
compatibility of the TANA dimers in a regular phosphodiester
backbone will be interesting and the work in this direction is
currently in progress.
8
9
10 T. Govindaraju and V. A. Kumar, Chem. Commun., 2005, 495–497;
T. Govindaraju and V. A. Kumar, Tetrahedron, 2006, 60, 2321–2330.
11 S. M. Frier and K.-H. Altmann, Nucleic Acids Res., 1997, 25,
This communication presents the results of selective RNA
recognition by a homogeneous DNA analogue and the ease of its
synthesis that has potential to be extended to purine nucleosides.
The thio function in the backbone may have an additional
advantage for better bioavailability of these modified oligodeoxy-
4
429–4443.
12 C. Thibaudeau, J. Plavec, N. Garg, A. Papchikhin and
J. Chattopadhyaya, J. Am. Chem. Soc., 1994, 116, 4038–4043.
3 M. Saneyoshi, T. Fujii, T. Kawagchi, K. Awai and S. Kimura, in
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New York, 1991, vol. 4, pp. 67–72.
14 K. J. Divakar and C. B. Reese, J. Chem. Soc., Perkin Trans. 1, 1982,
1
3
ribonucleosides. The simplicity of the approach described here is
1
171–1176.
equally appealing as is the selectivity.
1
5 S. K. Kim, P. E. Nielsen, M. Egholm, O. Buchardt, R. H. Berg and
B. Norden, J. Am. Chem. Soc., 1993, 115, 6477–6481.
16 P. Job, Ann. Chim., 1928, 9, 113–203.
K. G. thanks CSIR, New Delhi for a research fellowship.
V. A. K. thanks Director, NCL for financial assistance.
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Chem. Commun., 2006, 2373–2375 | 2375