A simple glycol nucleic acid

Article abstract of DOI:10.1021/ja042564z

A glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone forms stable antiparallel duplexes following the Watson-Crick base pairing rules. Copyright

Full text of DOI:10.1021/ja042564z

Published on Web 03/04/2005
A Simple Glycol Nucleic Acid
Lilu Zhang, Adam Peritz, and Eric Meggers*
Department of Chemistry, UniVersity of PennsylVania, 231 South 34th Street, Philadelphia, PennsylVania 19104
Received December 10, 2004; E-mail: meggers@sas.upenn.edu
We here wish to disclose a novel nucleic acid analogue which
displays exceptional structural simplicity and atom economy while
supporting stable duplex formation. The discovered glycol nucleic
acid (GNA) uses the canonical Watson-Crick base pairing scheme
combined with an acyclic three-carbon propylene glycol phos-
phodiester backbone (Figure 1).
Groundbreaking studies of the Eschenmoser group on the
chemical etiology of nucleic acid structure have demonstrated that
Watson-Crick base pairing can be supported by sugars in the
backbone that differ from RNA or DNA.1-3 Most surprisingly, a
nucleic acid derived from a tetrose sugar (TNA) was found to be
capable of antiparallel duplex formation and crosspairing with DNA
and RNA (Figure 1).4
Our laboratory is interested in structurally simplified nucleic acid
backbones in order to improve synthetic accessibility of artificial
duplexes. Inspired by Eschenmoser’s TNA structure, we envisioned
Figure 1. Comparison of the constitutions of DNA, RNA, and Eschen-
moser’s threofuranosyl nucleic acid (TNA) with our glycol nucleic acid
that acyclic glycol nucleosides could be synthesized just by
(GNA).
regioselective and stereospecific nucleophilic ring opening of a
“spring-loaded” epoxide.5,6
Accordingly, commercially available (R)-(+)- and (S)-(-)-
glycidol were tritylated to 1 following a standard protocol and
successfully ring opened with the unprotected nucleobases thymine
and adenine in the presence of substoichiometric amounts of NaH,
yielding 2 and 3, respectively (Figure 2).7 The thymine derivative
was directly transformed into the final phosphoramidite 4, whereas
the adenine derivative was first benzoylated at the exocyclic amino
group, followed by the formation of the final phosphoramidite 5
for the automated solid-phase oligonucleotide synthesis. With 4 and
5 in hand, we synthesized seven 18mer oligonucleotides consisting
entirely out of either enantiomer of the acyclic glycol nucleotides
(Table 1). (R)-(+)-glycidol yields (S)-GNA, and (S)-(-)-glycidol
yields (R)-GNA (Figure 1).
First, we investigated duplex formation of GNA with tempera-
Figure 2. Synthesis of glycol nucleotides for the automated solid-phase
ture-dependent UV spectroscopy at 260 nm. Figure 3 displays the
results with (S)-GNA. Mixtures (1:1) of two complementary strands,
6:7 and 8:9, yield characteristic sigmoidal melting curves with
melting points (TM) of 63 °C each, thus indicating cooperative
melting of GNA duplexes. No sigmoidal melting and weaker
hyperchromicities were observed with the single strands alone
(Figure 3a,b) or with two strands 8:10 that are complementary in
a parallel fashion (Figure 3c), indicating the requirement for
antiparallel duplex formation of GNA. To reassure that the duplex
relies on proper Watson-Crick base pairing, we also investigated
the UV melting behavior of an 18mer duplex 8:11 which contains
one T:T mismatch. The stability of this duplex decreases signifi-
cantly, yielding a TM of only 55 °C for the single mismatched
duplex. Furthermore, a duplex 8:12 with two mismatches (one T:T
and one A:A) is further destabilized with a TM of only 44 °C (Figure
3c). Identical results were obtained with the enantiomeric (R)-GNA
strands.
oligonucleotide synthesis. Shown is the route from (R)-(+)-glycidol leading
to (S)-GNA: (a) 0.2 equiv of NaH, DMF (49%). (b) (iPr2N)(NCCH2CH2O)-
PCl, 3 equiv of (iPr)2NEt (77%). (c) 0.2 equiv of NaH, DMF (51%). (d)
TMSCl, PhCOCl, then concentrated NH3 in H2O. (e) (iPr2N)(NCCH2CH2O)-
PCl, 3 equiv of (iPr)2NEt (52% over both steps).
enantiomers of GNA duplexes 8:9 and the individual single strands
are shown in Figure 4a and demonstrate strongly increased CD
bands at around 205, 220, and 275 nm upon mixing of 8 and 9 in
a 1:1 fashion, clearly supporting the formation of a helical duplex.
This CD signal is strongest at a 1:1 ratio of 8:9, as shown in the
insert of Figure 4a. In addition, the temperature-dependent CD
spectra in Figure 4b show that the Cotton effect decreases with
increasing temperature, again in agreement with the temperature-
dependent melting of a helical GNA duplex. As expected, the
enantiomeric (S)-GNA and (R)-GNA duplexes 8:9 yield mirror-
imaged CD spectra. Both spectra are very distinguished from a DNA
duplex of the same sequence (see Supporting Information).
The thermal stability of the GNA duplex 8:9 exceeds the stability
For subsequent analysis, we investigated duplex formation of
GNA with circular dichroism (CD). The CD spectra of both
of the analogous duplex of DNA (TM ) 40.5 °C) and RNA (TM
)
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10.1021/ja042564z CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Synthesized Sequences of 18mer GNA Oligonucleotides
3′-TTTTAAATTTTAATATAT-2′
2′-AAAATTTAAAATTATATA-3′
6
7
antiparallel duplex
(TM ) 63 °C)
3′-TAAAATTTATATTATTAA-2′
2′-ATTTTAAATATAATAATT-3′
8
9
antiparallel duplex
(TM ) 63 °C)
3′-TAAAATTTATATTATTAA-2′
3′-ATTTTAAATATAATAATT-2′
8
10
parallel duplex
(no TM)
3′-TAAAATTTATATTATTAA-2′
2′-ATTTTAAATTTAATAATT-3′
8
11
one mismatch
(TM ) 55 °C)
3′-TAAAATTTATATTATTAA-2′
2′-ATTTAAAATTTAATAATT-3′
8
12
two mismatches
(TM ) 44 °C)
Figure 4. (a) CD spectra of a 1:1 mixture of GNA strands 8 + 9 (4 µM
each), the individual strands 8 and 9 (4 µM each), and a mixture of glycol
nucleosides T and A (36 µM each) at 25 °C. Insert: CD signal at 220 nm
for different ratios of 8 to 9 in which the concentration of single strands 8
and 9 together is 8 µM for each data point. (b) Temperature-dependent CD
spectra of a 1:1 mixture (4 µM each) of GNA strands 8 + 9. Experiments
were performed in 10 mM sodium phosphate pH 7.0 with 200 mM NaCl.
The color coding is related to Figure 1.
forms highly stable antiparallel helical duplex structures following
the Watson-Crick base pairing rules. We believe that these
properties will render GNA a very valuable tool in contemporary
nucleic acid chemistry. At last, we wish to note that GNA may be
the most atom economical solution for a functional nucleic acid
backbone. We are therefore tempted to propose that GNA was a
potential predecessor of RNA as a genetic material and catalyst
for Earth’s earliest organisms.10
Figure 3. UV melting curves monitored at 260 nm. (a) (S)-GNA strands
6 + 7 (2 µM each) and the individual strands 6 (2 µM) and 7 (2 µM). (b)
(S)-GNA strands 8 + 9 (2 µM each) and the individual strands 8 and 9 (2
µM each). (c) (S)-GNA strands (2 µM each) 8 + 9 (leading to an antiparallel
duplex), 8 + 10 (parallel duplex), 8 + 11 (antiparallel duplex with one
mismatch), and 8 + 12 (antiparallel duplex with two mismatches). (d)
Duplexes with the sequence 8 + 9 (2 µM each strand) of RNA (U instead
of T), (S)-GNA, and RNA/(S)-GNA hybrid. Experiments were performed
in 10 mM sodium phosphate pH 7.0 with 200 mM NaCl, and 1 mM of
EDTA in experiments which include RNA strands. Data for UV melting
experiments were collected at 0.2 or 0.5 °C increments with a temperature
ramp of 0.5 °C/min.
Acknowledgment. We thank the University of Pennsylvania,
LRSM-MRSEC, and the ACS Petroleum Research Fund (Type G
grant) for supporting this research. We are also grateful for support
from the laboratories of Dr. Ivan J. Dmochowski (UV melting),
Dr. Marc M. Greenberg (UV melting), and Dr. Feng Gai (CD).
Supporting Information Available: Experimental procedures for
the synthesis of (S)-4, (R)-4, (S)-5, (R)-5, and their incorporation into
oligonucleotides, UV melting curves and CD spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
42.5 °C, U instead of T) by 22.5 and 20.5 °C, respectively. This is
astonishing considering the fact that the GNA backbone is
completely acyclic. It has been widely assumed that nucleic acid
analogues containing a phosphodiester backbone need to be cyclic
in order to produce the required conformational preorganization
for duplex formation.8,9 However, the GNA strands 8 and 9 show
a significant Cotton effect, suggesting a helical preorganization of
the GNA backbone already in the single strand. This conclusion is
also supported by the observation that a mixture of glycol
nucleosides T and A almost does not give any CD signals,
demonstrating that the Cotton effect is not simply the result of the
chiral centers in the glycol backbone (Figure 4a).
Finally, we investigated antiparallel crosspairing between (S)-
GNA, (R)-GNA, DNA, and RNA. We observed that (S)-GNA and
(R)-GNA do not undergo antiparallel crosspairing with each other,
nor do they form stable antiparallel crosspairs with DNA. On the
other hand, only (S)-GNA crosspairs with RNA (TM ) 35 °C for
RNA 8 with (S)-GNA 9; see Figure 3d). In this respect, it is
noteworthy that (S)-GNA is directly derived from TNA by
eliminating a CH2O unit from the tetrahydrofuran ring.
References
(1) For a review on the chemical etiology of nucleic acid structure, see:
Eschenmoser, A. Science 1999, 284, 2118-2124.
(2) For a review on oligonucleotide analogues with phosphodiester connectiv-
ity, see: Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841-854.
(3) For oligonucleotide analogues containing an uncharged pseudopeptide
backbone, see: (a) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624-630.
(b) Diederichsen, U. Angew. Chem., Int. Ed. Engl. 1997, 36, 1886-1889.
(4) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.;
Eschenmoser, A. Science 2000, 290, 1347-1351.
(5) For using the glycol backbone in a single metallo-nucleobase pair, see:
Zhang, L.; Meggers, E. J. Am. Chem. Soc. 2005, 127, 74-75.
(6) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004-2021.
(7) For related epoxide ring openings with nucleobases, see: (a) Baumgartner,
H.; Marschner, C.; Pucher, R.; Griengl, H. Tetrahedron Lett. 1991, 32,
611-614. (b) Ludek, O. R.; Meier, C. Synthesis 2003, 2101-2109.
(8) Acyclic nucleotides have been demonstrated to strongly destabilize duplex
DNA: (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990, 112,
453-455. (b) Peng, L.; Roth, H.-J. HelV. Chim. Acta 1997, 80, 1494-
1512.
(9) For the concept of conformational restriction of nucleosides as a measure
for preorganizing oligonucleotide single strands for duplex formation,
see: (a) Tarko¨y, M.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1993,
32, 1432-1434. (b) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1999,
121, 3249-3255. (c) Wengel, J. Acc. Chem. Res. 1999, 32, 301-310.
(10) For a review on the origin of life, see: Orgel, L. E. Trends Biochem. Sci.
1998, 23, 491-495.
In summary, we have described a structurally simple and
synthetically easily accessible acyclic glycol nucleic acid which
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