Effect of chirality of l/d-proline and prochiral glycine as the linker amino acid in five-atom linked thymidinyl-(α-amino acid)-thymidine dimers

Article abstract of DOI:10.1039/b913546d

The chirality of the amide linker in dimer blocks was found to have a profound effect on the orientation of base stacking interactions as studied by CD and NMR spectroscopy. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b913546d

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Effect of chirality of L/D-proline and prochiral glycine as the linker
amino acid in five-atom linked thymidinyl-(a-amino acid)-thymidine
dimersw
Seema Bagmare, Moneesha D’Costa and Vaijayanti A. Kumar*
Received (in Cambridge, UK) 7th July 2009, Accepted 8th September 2009
First published as an Advance Article on the web 25th September 2009
DOI: 10.1039/b913546d
The chirality of the amide linker in dimer blocks was found to
have a profound effect on the orientation of base stacking
interactions as studied by CD and NMR spectroscopy.
the binding strength of modified oligomers to target sequences.2b
In the thyminyl homo-oligomers recently reported by us,
L-amino acids were used alternating with thyminyl-b-amino
acids to form oligomers that recognized both DNA and
RNA.12 The implications of amino acid chirality in this
backbone are not yet studied. In the case of partial replace-
ment of phosphodiester linkages with the amide dimer units,
where it is necessary to keep the continuity of the backbone for
synergistic stacking interactions, such studies may be
important. To delineate structural differences, if any, that
L- or D-amino acids in the backbone would assert on the
geometry of the derived oligomers, T-(amino acid)-T dimers
are the simplest units where such effects can be investigated. In
this communication, we present synthesis of T-(amino acid)-T
dimer blocks with L-proline (Ia)/D-proline (Ib) and prochiral
glycine units (Ic) (Fig. 2). We chose to use the prochiral glycine
and L/D-prolines for our study, which can be extended to other
amino acids. L/D-Prolines were chosen because they are devoid
of amide N–H and hence remove the influence of hydrogen
bonds on the overall structural features.2h The effect of the
backbone chirality on the base stacking in the dimer building
blocks is studied by CD spectroscopy14 as reported earlier
for TpT,14a,b 20-O-methyl-TpT14e and LNA dimer blocks.15
Temperature-dependent NMR studies14b were carried out in
support of the CD results.
The constant quest for a simple, straightforward peptide
backbone superior to peptide nucleic acids (PNA)1 has been
a major research activity in our and other laboratories in the
last decade.2 The ever-increasing span of therapeutic inter-
ventions by oligonucleotides in antisense RNA,3 siRNA,4
miRNA,5 splice correction,6 etc. has further emphasized the
need for the development of modified oligonucleotides.
Because of its uncharged and achiral nature, PNA remains
hydrophobic and sparingly soluble in water. It also shows
aggregation properties and very low cell-uptake that necessitate
conjugation with cationic peptides or negatively charged DNA
to attain its optimum therapeutic value.7 Although PNA
posed quite a few problems for its application as a therapeutic
agent, the amide linker in the PNA backbone promised key
advantages over several other backbone modifications as
linkers in oligomers, most obvious being the applicability of
the well-established solid-phase peptide synthesis methodology.8
Over the last decade, we and others have been working
towards improving PNA properties by introduction of chirality,9
positive charges,10 peptide conjugation,11 etc. with remarkable
success in terms of novelty and creativity. The very recent and
striking example is that of prolyl-(ACPC)-PNA consisting of
alternating a-amino acid proline as a nucleobase carrier and
chiral b-aminocyclopentane carboxylic acid.2h We later
reported a similar concept but a synthetically more straight-
forward (a- + b-) amino acid backbone in which the b-amino
acid derived from thymidine alternated with naturally
occurring a-L-amino acids such as proline, sarcosine, lysine
and methionine, yielding amide-linked oligomers12 (Fig. 1). It
is known that in DNA, the phosphate group connecting the
two nucleosides is prochiral and when rendered chiral due to
substitution at phosphorus, affects DNA/RNA recognition
processes.13 The partial replacement of the DNA/RNA
phosphodiester linker by five-atom amide linkers has been
found to be very useful for RNA recognition.2b,c Also, in
earlier studies, chirality of amide linkers was found to affect
The synthesis of the dimeric building blocks Ia using
L-proline as
a representative example is depicted in
Scheme 1. 50-Dimethoxytrityl-30-deoxy-30-aminothymidine 1
was synthesized by literature-reported procedures.2c Acylation
of 1 using Fmoc-protected L-proline (2a), D-proline (2b) or
glycine (2c) yielded 3a, 3b and 3c, respectively. The inter-
mediate free amino compounds 4a, 4b and 4c obtained
by removing the Fmoc protecting group in 3a/b/c were
N-acylated using 30-O-tert-butyldimethylsilylthymidine-40-
carboxylic acid 5. Compound 5 was obtained by TEMPO–BAIB
Division of Organic Chemistry, National Chemical Laboratory,
Pune 411008, India. E-mail: va.kumar@ncl.res.in;
Fax: +91 20 25902629; Tel: +91 20 25902340
w Electronic supplementary information (ESI) available: 1H NMR,
mass spectral data for compounds Ia, Ib, Ic, N/S conformational
analysis of Ia, Ib, Ic, temperature dependent change in H-6 H NMR
1
signals. See DOI: 10.1039/b913546d
Fig. 1 DNA, RNA and modified PNA analogues.
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Fig. 2 Proposed dimer blocks containing L-Pro Ia, D-Pro Ib and
glycine Ic as linker amino acids.
oxidation of the 50-alcohol of 30-O-tert-butyldimethylsilyl-
thymidine using a reported procedure.16 The individual
products 6a, 6b and 6c obtained after desilylation using TBAF
in dry THF, were purified by silica-gel column chromato-
graphy and characterized using NMR and mass spectral
analyses. For the present studies 6a, 6b, 6c were detritylated
to give Ia, Ib, Ic, respectively. All the three dimer units were
extensively characterized by NMR, and confirmed by mass
spectral analysis. The 1H assignments were confirmed by
2D-COSY studies. a-Amino acid peptides prefer to be trans
oriented with respect to the amide bond because of steric
reasons and the Z : E ratio is about 1000 : 1 except in the case
of proline. In prolines, the Z-form is only slightly favored
over the E-form because of steric interactions involving the
o-carbon of the proline ring.17 This preference was also
observed in the case of proline-linked nucleoside dimer building
blocks Ia and Ib. The remote sensing of trans/cis isomers at the
prolyl amide bond is evident from the major : minor (83 : 17 in
Ia and 87 : 13 in Ib) H10 NMR signals corresponding to the
50- and 30-thyminyl units in dimers where the internucleoside
amino acid linker is either L- or D-proline. This difference is
absent in Ic where glycine is used as the internucleoside amino
acid. We further studied the conformational preferences of the
sugar residues in the dimer units by using the Sum rule
(ESIw).18 In all the three dimers Ia, Ib and Ic, the 30-sugar
exhibited higher N-type geometry uniformly as compared to
the 50-thyminyl units for the major isomer. The percentage of
N character was found to be higher where sugar ring is
substituted by nitrogen in the ring B (50–64%) as compared
to 30-OH sugar (18–28%) in ring A.
Scheme 1 Synthesis of dimer building blocks.
and Ic to evaluate any contributions of chirality of the
a-amino acid towards stacking interactions. Indeed, the
chirality of the amino acid in the dimers was found to exert
profound effects on the CD signals. The D-proline containing
T-(D-proline)-T Ib showed a strong positive band at 275 nm
[Fig. 3(a)] and a negative band at 240 nm. In the T-(L-proline)-
T dimer Ia, along with the negative band at 240 nm, two
distinct low intensity CD bands were observed at 265 and
290 nm along with a broad minimum at 275–280 nm
[Fig. 3(a)]. The T-(glycine)-T dimer Ic showed low intensity
broad bands at 240 and 280 nm [Fig. 4(c)]. The high intensity
of CD signal at B280 nm leads us to believe that Ib is well
stacked even at the dimer level and the orientation of stacked
species is similar to the LNA dimer blocks.15 The minimum
at B275 nm in Ia is comparable to the negative CD signal
at B275 nm in the case of the homogeneous amide-backbone
T-[(L-pro-T)]7 octamer [Fig. 3(b)] earlier reported by us.12
Thus, the stacking interactions in Ia and Ib could be arising
from different adopted conformations due to opposite
chirality in the internucleoside linker giving rise to opposite
CD signals at B275 nm. Glycine, being prochiral, could either
result in the existence of both chiral components in equal
measure, which cancel each other, or may not contribute to the
base-stacked structure, and eventually lead to only a weak
maximum at B280 nm. To our knowledge, this is the
The chirality of the internucleoside linker such as prochiral
natural phosphate, Rp and Sp chiral phosphorothioates13 or
R/S amides2b is known to exert large effects on the binding
efficiency of the oligomers but the origin of this binding
discrimination has not been studied on the molecular level.
In these cases also the sugar conformations do not drastically
differ in the diastereomers formed because of the chirality of
the internucleoside linker as in the case of the dimers Ia, Ib and
Ic. Conversely, the N-type conformational preference of the
sugar ring in 20-OMe or LNA type oligomers is known to
dramatically improve the binding efficiency of the oligomers
and in these cases, effective base stacking is one of the
well-established contributing factors to the duplex stability.
Increase in the positive CD band at 275 nm is ascribed to the
effective base stacking interactions even at the dimer level.15
We therefore carried out CD studies on the dimer blocks Ia, Ib
Fig. 3 (a) CD spectra of T-(L-Pro)-T Ia (blue) and T-(D-Pro)-T Ib
(red) at a concentration of 100 mM in water; (b) CD spectrum of
T-[(L-Pro)-T]7.12
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Fig. 4 Temperature-dependent CD studies of thymine dimers containing (a) L-proline Ia, (b) D-proline Ib and (c) glycine Ic at 100 mM
concentration, in water at 10 (red), 20 (black), 40 (blue), 60 (light blue) and 80 1C (purple).
first report where such profound effects of backbone linker
chirality on the resulting dimer CD signals are reported.
The strong positive band at B275 nm in the T-(D-proline)-T
dimer Ib displayed significant temperature dependence from
10–80 1C [Fig. 4(b)] as reported earlier for the TpT dimer
block.14b For the T-(L-proline)-T dimer Ia, increasing
temperature resulted in the reduction of the amplitude of
CD minimum band at B275 nm [Fig. 4(a)]. These changes
in intensities of CD maxima and minima imply the presence of
higher fractions of stacked species in Ib and Ia. No temperature-
dependent change in CD intensity was observed in the case of
T-(glycine)-T dimer Ic [Fig. 4(c)] and thus fractions of stacked
species are less significant at the dimer level in Ic. The
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S. B. thanks UGC, New Delhi for a research fellowship.
V. A. K. thanks Wellcome Trust, UK and CSIR, New Delhi
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This journal is The Royal Society of Chemistry 2009
6648 | Chem. Commun., 2009, 6646–6648