Synthesis and Stability of Oligonucleotides Containing Acyclic Achiral Nucleoside Analogues with Two Base Moieties

Article abstract of DOI:10.1021/ol0360647

(Equation presented) Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting sequence information at the outside of the helix.

Full text of DOI:10.1021/ol0360647

ORGANIC
LETTERS
2004
Vol. 6, No. 1
51-54
Synthesis and Stability of
Oligonucleotides Containing Acyclic
Achiral Nucleoside Analogues with Two
Base Moieties
Tongfei Wu, Matheus Froeyen, Guy Schepers, Kristof Mullens, Jef Rozenski,
Roger Busson, Arthur Van Aerschot, and Piet Herdewijn*
Laboratory of Medicinal Chemistry, Rega Institute for Medicinal Research,
Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, B-3000 LeuVen, Belgium
piet.herdewijn@rega.kuleuVen.ac.be
Received October 22, 2003
ABSTRACT
Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved
in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting
sequence information at the outside of the helix.
The double-stranded DNA consists of two phosphorylated
deoxyribose backbones on which purine and pyrimidine
bases are attached, base pairing with each other. The
sequence of bases constitutes the informational part of DNA.
As demonstrated by Watson and Crick,1 this genetic infor-
mation is pointed inward to the double helix and unwinding
is needed to make this information available (for replication
and transcription). However, for building less complicated
systems (compared to the biological organization of a cell),
it would be more straightforward when this information can
be used more directly, i.e., when the bases are pointing
outside the helix. DNA consists of three moieties (phosphate,
sugar, base), and all three of them can be used for presenting
information outside the helix. The internucleotide phosphate
group has been used intensively to attach reporter groups2
or cationic residues3 or to be replaced by other information-
containing functionalities.4 Also the sugar moiety has been
presented outside the helix structure by using disaccharide
nucleotide units.5 Here, we present the synthesis of a double-
headed oligonucleotide that can be used to present sequence
information at the outside of the double helix (Figure 1).
(2) (a) Agrawal, S. Methods Mol. Biol. 1994, 26, 93. (b) Fidanza, J. A.;
Ozaki, H.; Mclaughlin, L. W. Methods Mol. Biol. 1994, 26, 121.
(3) Michel, T.; Debart, F.; Vasseur, J.-J. Tetrahedron Lett. 2003, 44,
6579.
(4) Vandendriessche, F.; Van Aerschot, A.; Voortmans, M.; Janssen, G.;
Busson, R.; Van Overbeke, A.; Van Bossche, W.; Hoogmartens, J.;
Herdewijn, P. J. Chem. Soc., Perkin Trans. 1 1993, 1567.
(5) Nauwelaerts, K.; Vastmans, K.; Froeyen, M.; Kempeneers, V.;
Rozenski, J.; Rosemeyer, H.; Van Aerschot, A.; Busson, R.; Lacey, J. C.;
Efimtseva, E.; Mikhailov, S.; Lescrinier, E.; Herdewijn, P. Nucleic Acids
Res. 2003, in press.
(1) Watson, J. D.; Crick, F. H. Nature 1953, 171, 737.
10.1021/ol0360647 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/05/2003

Scheme 1. Synthesis of Nucleoside Phosphoramidite Building
Blocks
Figure 1. Picture of the Watson-Crick double helix (A) and of a
duplex containing double-headed DNA (B).
The synthesis and hybridization properties of the first
example of double-headed DNA are described.
Oligonucleotides containing acyclic nucleoside analogues
usually bind poorly to DNA and RNA as a result of loss in
entropy upon duplex formation, caused by the flexibility of
the acyclic units.6 Despite this, we preferred to use an acyclic
nucleotide as our first model. Indeed, we do not have
information on the geometry that is needed for a nucleotide
containing two base moieties to fit in a helix structure. To
start with a simple model, we also decided to synthesize a
symmetric nucleoside building block (i.e., with identical
bases) having two primary hydroxyl groups (to avoid much
steric hindrance for the tritylation and phosphoramidite
synthesis and to avoid the chirality problem). This is
exemplified by the nucleosides 2,2-bis[(thymin-1-yl)methyl]-
propane-1,3-diol (T*) and 2,2-bis[(adenin-9-yl)methyl]-
propane-1,3-diol (A*).
The synthetic route to 1-O-monomethoxytrityl-2,2-bis-
[(thymin-1-yl)methyl]-1,3- propanediol phosphoramidite 6a
and 1-O-monomethoxytrityl-2,2-bis[(adenin-9-yl)methyl]-
1,3-propanediol phosphoramidite 6b is outlined in Scheme
1. The 2-phenyl-5,5-bis(hydroxyl methyl)-1,3-dioxane 1a was
synthesized according to the procedure described previously.7
Treatment of 1a under Mitsunobu conditions with Ph3P, N3-
benzoylthymine, and diethyl azodicarboxylate (DEAD) in
THF offered 2-phenyl-5,5-bis(thymin-1-yl)-1,3-dioxane 2a
in 85% yield. The benzylidene protecting group of 2a was
removed using TFA/H2O (3/1), giving 3a in 90% yield,
followed by tritylation with MMTrCl in dry pyridine to afford
4a in 56% yield. Removal of the benzoyl protecting group
was achieved by treatment of compound 4a with saturated
NH3/MeOH to give 5a in 70% yield. Finally, phosphitylation
of compound 5a gave the desired phosphoramidite 6a in 80%
yield.
adenine using NaH and adenine in dry DMF, giving 2b in
48% yield. Protection of the exocyclic amino group of the
adenine base was achieved using excess benzoyl chloride in
pyridine. Subsequent treatment with saturated NH3/MeOH
at 0 °C for 30 min resulted in the desired monobenzoyl-
protected amines 3b in 56% yield. Removal of the ben-
zylidene group of 3b by treatment with TFA/H2O (3/1)
afforded 4b in 93% yield, which was reacted with MMTrCl
in anhydrous pyridine to give 5b in 55% yield. Finally,
phosphitylation of compound 5b gave the desired phos-
phoramidite 6b, in a somewhat lower yield of 52%.
With the phosphoramidites 6a and 6b of the building
blocks T* and A* at hand, several modified oligonucleotides
were synthesized comprising single or multiple bis-thyminyl
or bis-adeninyl nucleotides. The thermal stability of these
oligonucleotides with either DNA or RNA as complement
was investigated. The results for the influence of incorpora-
tion of one modified T* and one modified A* on duplex
stability of a mixed dsDNA sequence are shown in Table 1.
Likewise, the influence of mismatches opposite T* and A*
on the thermal stability of the same duplex is shown.
Compared to unmodified DNA, incorporation of the
modified nucleotide T* or A* in the middle of the oligo-
nucleotides led to a decrease in Tm of 5.9 or 8.0 °C,
respectively (entries 2 and 7). At first sight, this decrease in
Compound 1a was converted to 1b as described previ-
ously.8 The two tosyloxy groups of 1b were substituted with
(6) (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990, 112,
453. (b) Augustyns, K.; Van Aerschot, A.; Van Schepdael, A.; Urbanke,
C.; Herdewijn, P. Nucleic Acids Res. 1991, 19, 2587. (c) Vandendriessche,
F.; Augustyns, K.; Van Aerschot, A.; Busson, R.; Hoogmartens, J.;
Herdewijn, P. Tetrahedron 1993, 49, 7223. (d) Boesen, T.; Pedersen, D.
S.; Nielsen, B. M.; Petersen, A. B.; Henriksen, U.; Dahl, B. M.; Dahl O.
Bioorg. Med. Chem. Lett. 2003, 13, 847. (e) Nielsen, P.; Dreiøe, L. H.;
Wengel, J. Bioorg. Med. Chem. Lett. 1995, 3, 19.
(7) Lakshmi, B.; Devi, A. P.; Nagarajan, M. J. Chem. Soc., Perkin Trans.
1 1997, 10, 1495.
(8) Wang, Q.; Mikkola, S.; Loennberg, H. Tetrahedron Lett. 2001, 42,
2735.
52
Org. Lett., Vol. 6, No. 1, 2004

Table 1. Influence of Incorporation of One Modified
Nucleotide (A* or T*) on dsDNA Stability and Their Mismatch
Discrimination Properties
sequences d(3′-GGTCACXATACG-5′)
d(5′-CCAGTGYTATGC-3′)
X-Y
Tm
∆Tm
X-Y
Tm
∆Tm
entry base pair (°C)a (°C) entry base pair (°C)a (°C)
1
2
3
4
5
T-A
49.1
43.2
34.1 -15.0
36.0 -13.1
29.1 -20.0
6
7
8
9
10
A -T
A*-T
A*-A
A*-G
A*-C
49.4
41.4
35.3 -14.1
36.3 -13.1
31.3 -18.1
T*-A
T*-T
T*-G
T*-C
-5.9
-8.0
a Tm was determined at UV 260 nm in NaCl (0.1 M), KH2PO4 (20 mM,
pH 7.5), EDTA (0.1 mM), with 4 µM of each strand.
Figure 2. The average structure obtained from the simulation. The
average structure was calculated from the last 50 ps of a 500-ps
MD simulation. Figure created with Molscript.
melting point may lead to the conclusion that no base pairing
occurs at the modified site. The mismatch sequences are
considerably less stable, however, and a ∆Tm ranging from
-13.1 to -20.0 °C is observed. This means that base pair
discrimination is still present and that the T-A base pair is
more stable than the mismatches.
To visualize the potential interaction of the modified
nucleotide at the insertion site, a model was built. The duplex
used for model building has the sequence 5′-(GCATAT*-
CACTGG)-3′ with its complement, with T* representing the
modified nucleotide. The final 50 ps were used to calculate
an average structure (Figure 2).
A hydrogen bond analysis does not give an indication of
broken or missing hydrogen bonds in the duplex. However,
the higher flexibility of the T* residue might destabilize the
duplex and act as a nucleus of disorder in the duplex when
heating the system, resulting in a lower melting temperature
compared to that of the unmodified duplex.
The hybridization properties of oligonucleotides, contain-
ing more than one successive modification, toward comple-
mentary DNA are shown in Table 2. This experiment is
carried out with the aim of determining if the presence of
more sequence information outside the helix will not lead
to unacceptable duplex destabilization.
binding affinity. However, the ∆Tm mod-1 decreases going
from one to four incorporated A* moieties (incorporation
of one A* causes relative more destabilization than three or
four A* ∆Tm mod-1, which is demonstrated with different
sequences). This might suggest that some stacking interaction
between the extra helical adenine bases of A* is possible. It
should be mentioned that this effect is not seen with T*,
i.e., a pyrimidine base that is known to demonstrate less
stacking properties and that gives more steric hindrance at
the N1 position, giving a pyrimidine base less opportunity
for rotation around the C1′-N1 bond.
Hybridization of modified oligonucleotides with comple-
mentary RNA was studied likewise (Table 3). For oligo-
nucleotides containing modified thymine or adenine nucle-
otides (T* or A*), the ∆Tm mod-1 decreases as the number
of modified thymidine or adenine nucleotides (T* or A*)
increases (up to five T* and three A* were evaluated). The
oligonucleotide with three successive T* displayed a Tm of
40.8 °C (entry 20). Upon incorporation of five T* monomers,
the Tm (entry 21) versus the RNA complement increased to
47.7 °C, almost identical to the Tm of the duplex with only
one modified T* modification (entry 19). Notably, the Tm
Increasing the number of A* nucleotides within an
oligonucleotide results in further reduction of absolute
Table 2. Influence of Incorporation of Several Modified Nucleotides (A*) on the Stability of Deoxyoligonucleotides Hybridized to
Their DNA Complement
entry
sequences
Tm (°C)a
∆Tm mod-1 (°C)
mass calcdb
ESI-MS
11
12
13
14
15
16
17
d(5′-CGGCAAAAACGCC-3′)
59.1
50.8
43.9
60.8
49.3
44.7
36.3
d(5′-CGGCAAA*AACGCC-3′)
d(5′-CGGCAA*A*A*ACGCC-3′)
d(5′-CGGCAAAAAACGCC-3′)
d(5′-CGGCAAA*A*AACGCC-3′)
d(5′-CGGCAA*A*A*A*ACGCC-3′)
d(5′-CGGCA*A*A*A*A*A*CGCC-3′)
-8.3
-5.1
4054.8
4292.9
4055.3
4293.4
-5.7
-4.0
-4.0
4486.9
4725.0
4963.1
4487.3
4725.5
4963.7
a Tm was determined at UV 260 nm in NaCl (0.1 M), KH2PO4 (20 mM, pH 7.5), EDTA (0.1 mM), with 4 µM of each strand. b Calculated monoisotopic
mass.
Org. Lett., Vol. 6, No. 1, 2004
53

Table 3. Influence of Incorporation of Several Modified Nucleotides (A* or T*) on the Stability of Deoxyoligonucleotides Hybridized
to Their RNA Complement
entry
sequences
Tm (°C)a
∆Tm mod-1 (°C)
mass calcdb
ESI-MS
18
19
20
21
22
23
24
d(5′-GGCGTTTTTGCCG-3′)
d(5′-GGCGTTT*TTGCCG-3′)
d(5′-GGCGTT*T*T*TGCCG-3′)
d(5′-GGCGT*T*T*T*T*GCCG-3′)
d-AAAAAAAAAAAAA
56.7
48.7
40.8
47.7
14.5
13.3
15.1
-8.0
-5.3
-1.8
4080.7
4300.8
4520.9
4081.2
4301.3
4521.4
d-AAAAAAA*AAAAAA
d-AAAAAA*A*A*AAAAA
-1.2
+0.2
4126.9
4365.0
4127.6
4365.9
a Tm was determined at UV 260 nm in NaCl (0.1 M), KH2PO4 (20 mM, pH 7.5), EDTA (0.1 mM), with 4 µM of each strand. b Calculated monoisotopic
mass.
for the oligonucleotide with three A* modifications is 0.6
propane- 1,3-diol were likewise synthesized. The duplex with
complementary DNA or RNA proved unstable.
°C higher than for the unmodified duplex (entry 24), although
we deal here with a different model [oligo (dA): oligo U] of
low stability.
Overall we could demonstrate that stable double-stranded
DNA can be obtained presenting base moieties outside the
helix. These results encourage us to evaluate in the future
the stability of new double-headed DNA with the same and
different bases attached to a sugar or sugarlike motif instead
of the acyclic motif and to investigate the way this informa-
tion can be used.
In conclusion, novel oligonucleotides containing 2,2-bis-
[(thymin-1-yl)methyl]-propane-1,3-diol and 2,2-bis[(adenin-
9-yl)methyl]-propane-1,3-diol were assembled on an auto-
mated DNA synthesizer. Oligonucleotides with one acyclic
achiral nucleoside (A* or T*) incorporated in the middle of
a 12-mer or 13-mer show decreased hybridization properties
with complementary DNA or RNA. Likewise, oligonucle-
otides comprising several consecutive acyclic achiral nucleo-
sides (A*) in the middle show pronounced reduction in
duplex stability for their complementary DNA. The ∆Tm
mod-1, however, decreases with the incorporation of suc-
cessive modification. Using RNA as the complement, the
duplexes are much more stable, and in some cases, even an
increase in duplex stability is observed. Acyclic oligonucle-
otides consisting entirely of 2,2-bis[(thymin-1-yl)methyl]-
Acknowledgment. The research was supported by a grant
from the K.U.Leuven (GOA 2002/13). The authors thank
Chantal Biernaux for editorial help.
Supporting Information Available: Experimental pro-
cedures and spectral data for compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0360647
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Org. Lett., Vol. 6, No. 1, 2004