Chimeric (α-amino acid + nucleoside-β-amino acid)n peptide oligomers show sequence specific DNA/RNA recognition

Article abstract of DOI:10.1039/b716835g

An α/β-peptide backbone oligonucleotide comprising natural α-amino acids alternating with a β-amino acid component derived from thymidine sequence specifically recognizes and binds to deoxy- and ribo-oligoadenylates in triplex mode. The Royal Society of Chemistry.

Full text of DOI:10.1039/b716835g

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Chimeric (a-amino acid + nucleoside-b-amino acid)n peptide oligomers
show sequence specific DNA/RNA recognition{
Khirud Gogoi* and Vaijayanti A. Kumar*
Received (in Cambridge, UK) 31st October 2007, Accepted 27th November 2007
First published as an Advance Article on the web 12th December 2007
DOI: 10.1039/b716835g
The prolyl-ACPC backbone seems very interesting as this
alternating a/b amino acid backbone exhibited preferential binding
with complementary DNA but not with complementary RNA
sequences. We perceived that alternating nucleoside-b-amino acids
with natural a-amino acids (Fig. 2) would give rise to similar
alternating a/b amino acid backbone scaffolds. A literature search
revealed that similar work on phosphate free oligonucleoside
analogues is reported in a patent.17 The internucleoside amino
acids used were only glycine and lysine in that study and only
binding studies with complementary DNA oligomers were
reported. In the light of recent developments in this area, we
thought it worthwhile to reconstitute the sugar–amino acid
backbone incorporating different natural amino acids for RNA
binding studies.
An a/b-peptide backbone oligonucleotide comprising natural
a-amino acids alternating with a b-amino acid component
derived from thymidine sequence specifically recognizes and
binds to deoxy- and ribo-oligoadenylates in triplex mode.
Novel oligonucleotide (ON) analogues that can form stable
duplexes or triplexes with target nucleic acids (NA) are important
synthetic objectives because of their use as therapeutic agents in
antisense and antigene strategies.1 Various types of modified
oligonucleotides have been developed over the last two decades as
potential diagnostic probes and antisense and antigene therapeu-
tics.2 The more recent developments such as splice correcting3 and
exon skipping3 strategies require highly robust nucleic acid
analogues that are stable under physiological conditions as single
strands as well as in the form of duplexes with complementary
RNA sequences. The study of unnatural de-phosphono nucleic
acid oligomers is gaining in importance for such applications. The
replacement of the internucleoside sugar–phosphate linkages by
the robust sugar–amide bond could be advantageous as in PNA.4,5
This would maintain the chirality and 39–59 directionality of the
backbone, along with the enzymatic stability of the amide bond.
Several four- and five-atom amide-linked (6/7 atom repeating
units) deoxy6,7 and ribo8,9 ON analogues are known in the
literature. The other relevant examples of 6/7-atom repeating units
are those of pyrrolidine-based NA analogues10 such as pyrrolidi-
nyl11/POM-PNA12 and bepPNA (Fig. 1)13 or those comprising
prolyl-aminopyrrolidine-2-carboxylic acid14 and prolyl-2-aminocy-
clopentanecarboxylic acid (prolyl-ACPC backbone) (Fig. 1).15,16
In this communication, we report the synthesis and DNA/RNA
binding studies of amide linked oligothyminyl DNA analogues
(Fig. 1) comprising conformationally constrained a-amino acids
(L-proline 2 and sarcosine 3, Fig. 2), a positively charged a-amino
acid (L-lysine 4, Fig. 2) and a neutral a-amino acid (L-methionine
5, Fig. 2) in the backbone, alternating with a b-amino acid
nucleoside component derived from thymidine (1, Fig. 2). The
potential advantages are as follows. (a) In 39-deoxy-39-amino-
29-deoxyribose sugar the five-membered ring pucker is in a
preferred 39-endo conformation that is better suited for m-RNA
recognition.18 (b) The new synthesis of nucleoside-b-amino acid is
quite simple using a recently reported TEMPO–BAIB method.19
(c) The flexibility of the five atom internucleoside amide linker
could be better fitting for replacement of a phosphate group for
RNA binding taking into account the shorter amide bond
compared to the phosphodiester linkage.7 (d) A negatively charged
phosphate group can be replaced by neutral (proline, sarcosine and
methionine) or positively charged20 (lysine) alternatives.
Our synthetic strategy was aimed at assembling these molecules
on a solid support, for its subsequent adaptation to well
established conventional peptide chemistry.21 Oxidation of the
59-hydroxy moiety of 39-azidothymidine 6 to obtain 7 was reported
earlier17,22a by Varma et al. using RuCl3–Na2S2O8–KOH. The use
of strongly alkaline conditions in this reaction makes it unsuitable
Fig. 1 Structure of DNA/RNA and amino acid backbone modified
ONs.
Division of Organic Chemistry, National Chemical Laboratory, Pune,
411008, India. E-mail: k.gogoi@ncl.res.in; va.kumar@ncl.res.in;
Fax: +91-20-25902624; Tel: +91-20-25902340
{ Electronic supplementary information (ESI) available: Experimental
procedures; H, 13C, mass spectra of compounds 7 and 8; MALDI-TOF
mass spectra of ONs 12–15; UV melting experiments and CD curves. See
DOI: 10.1039/b716835g
1
Fig. 2 Structures of monomer units.
706 | Chem. Commun., 2008, 706–708
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a
Table 1 UV-Tm values (in uC) of chimeric modified
ONs:complementary DNA/RNA
No. Sequence
DNA 9 RNA 10 DTmRNA–DNA
1
2
3
4
5
a
TTTTTTTT 11 17.8
b-Ala-(Pro-t)8-H 12 49.0
15.6
60.8
61.6
69.0b
57.3
—
+11.8
+12.5
+12.7
+10.0
b-Ala-(Sar-t)8-H 13
(Lys-t)8-H 14
b-Ala-(met-t)8-H 15 47.3
49.1
56.3b
Scheme 1 Synthesis of thyminyl sugar amino acid monomer unit.
Reagents and conditions: (a) TEMPO–BAIB, acetonitrile–water; (b) Pd/C–
H2, methanol; (c) Fmoc-succinimide, NaHCO3, acetone–water. TEMPO
2,2,6,6-tetramethyl-1-piperidinyloxyl; BAIB [bis(acetoxy)-iodo]benzene;
Fmoc fluorenylmethoxycarbonyl.
Tm 5 melting temperature measured in 10 mM sodium phosphate
buffer, pH 7.0 with 100 mM NaCl and 0.1 mM EDTA, from 5 to
85 uC at ramp 0.5 uC. All values are an average of three independent
experiments and accurate to within ¡0.5 uC. T denotes DNA
backbone. t denotes amide backbone. DNA 9: 59 GCAAAAA-
AAACG 39, RNA 10: r(59 GCAAAAAAAACG 39). UV melting
experiments were performed at pH 5.5 also but no significant change
in melting temperatures was detected.
b
for adaptation to other amino-protected nucleosides. The products
were also often contaminated with inorganic salts. Oxidation using
Sharpless conditions works well only for purine nucleosides and
not with pyrimidine nucleosides.22b A recently published TEMPO–
BAIB method19a used for 29,39-isopropylidene ribonucleosides
could be a more general procedure for nucleosides19b as it is
reported to be compatible with several protecting groups and other
functionalities such as azide. The products isolated are not
contaminated with any inorganic salts. Thus, 6 was easily
converted to the corresponding acid 7 under TEMPO–BAIB
oxidation conditions and the product was recovered by trituration
with ether and acetone (Scheme 1). The 39-azido group in 7 was
then hydrogenated using Pd–C to an amine which was
subsequently Fmoc protected to give the monomer building block
8. The monomer 8 along with protected a-L-amino acids (2, 3, 4
and 5) was used to synthesize four octameric NA sequences (12,
13, 14 and 15) on a rink amide resin support using an Fmoc
peptide synthesis strategy. For sequences 12, 13 and 15, b-alanine
was used as a linker to the resin and for sequence 14 no linker was
used.23 All the coupling reactions were monitored by the Kaiser
test. The oligomers were cleaved from the solid support using
standard conditions and were purified by gel-filtration followed by
RP-HPLC. Finally the purity and integrity of the oligomers were
established by MALDI-TOF mass spectrometry (ESI{).
(Tm) higher than those of the complexes formed by oligothymi-
dinyl-DNA fragment 11 of the same length (Fig. 3, Table 1).
Complexes formed by all the modified oligomers 12, 13, 14 and 15
with the RNA target showed higher stability than similar
complexes formed with the DNA target (Table 1). The positively
charged lysine oligomer 14 formed the most stable complexes with
both DNA (14:9) and RNA (14:10). The UV melting experiments
of ON 14 with complementary DNA 9 and RNA 10 were
performed at pH 5.5 but no significant change in melting
temperature could be detected, indicating that the side chain lysine
amine group could be protonated even at physiological pH. A
single base mismatch caused a fall in Tm (11–18 uC) in the case of
either DNA or RNA targets (ESI{) similar to the case of PNA.4c
A UV–Job’s plot of the modified oligomers with complementary
DNA and RNA clearly showed a 2 : 1 stoichiometry of the
complex (ESI{).
The CD spectra of single strands 12–15 showed ellipticity
minima at y280 nm with no positive CD band (ESI{). The CD
spectrum of the 2 : 1 hybrid formed between 12 and complement-
ary RNA 10 showed ellipticity minima at 246 nm and ellipticity
maxima at 260 and 281 nm (Fig. 4). The CD spectra of the
complexes of 13, 14 and 15 with complementary RNA showed a
similar CD signature (Fig. 4), substantiating the formation of triple
helical structures.25
The hybridization properties of the amino acid modified
oligomers 12, 13, 14 and 15 with complementary DNA or RNA
ONs GCAAAAAAAACG 9 or r(GCAAAAAAAACG) 10 were
determined by thermal denaturation studies.24 UV melting
experiments indicate that oligomers 12, 13, 14 and 15 hybridized
to complementary DNA and RNA with melting temperatures
Thus, the complexes between chimeric (a-amino acid + nucleo-
side-b-amino acid) backbone ONs (12, 13, 14 and 15) and their
Fig. 3 UV melting graph of ONs 12, 13, 14 and 15 with RNA 10 (in
10 mM sodium phosphate buffer, pH = 7.0, 100 mM NaCl, and 0.1 mM
EDTA). RNA 10: r(59 GCAAAAAAAACG 39).
Fig. 4 CD graph of ONs 12, 13, 14 and 15 with RNA 10 (in 10 mM
sodium phosphate buffer, pH 5 7.0, 100 mM NaCl, and 0.1 mM EDTA).
RNA 10: r(59 GCAAAAAAAACG 39).
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complementary DNA/RNA (9/10) oligonucleotides were found to
be highly stable as compared to DNA:DNA (11:9) or DNA:RNA
(11:10) duplexes. The presence of a positively charged a-amino
acid (L-lysine) contributes further to the strong binding properties
(7–8 uC). The stability of complexes of sarcosine and proline
analogues, similar to the prolyl-ACPC PNA, in which one amide
hydrogen is absent, suggests the absence of a contribution from the
known secondary structures of the (a-amino acid + b-amino acid)
peptide scaffolds26 to the overall stability of the complexes. The
5-atom amide linked oligomers in this simple (a-amino acid +
nucleoside-b-amino acid) backbone exhibit much higher stability
of complexes with both complementary DNA and RNA,
complexation with RNA being favoured over DNA. The basis
of this selectivity could be similar to that for the other 5-atom
internucleoside amide linkages.7,27
The use of naturally occurring a-amino acids in conjunction
with an easily accessible nucleoside derived b-amino acid described
in this communication thus provides a very simple peptide scaffold
to create a nucleobase sequence for DNA/RNA recognition.
Further studies on the mixed purine–pyrimidine sequences as well
as incorporation of negatively charged and D-amino acids into the
backbone to study the stereoelectronic requirements of the
complexes are currently in progress in our laboratory.
KG thanks CSIR, New Delhi for a senior research fellowship.
VAK thanks the Department of Science and Technology, New
Delhi and the Director, National Chemical Laboratory for
research grants.
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708 | Chem. Commun., 2008, 706–708
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