Preparation and properties of a new type of acyclic, achiral nucleoside analogue

Article abstract of DOI:10.1081/NCN-120021967

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by T_m measurements showing a ΔAT_m of -2 to -6.5°C per modification. An oligonucleotide with two modifications at the 3′-end showed considerable resistance towards cleavage by a 3′-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.

Full text of DOI:10.1081/NCN-120021967

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 623–627, 2003
Preparation and Properties of a New Type of Acyclic,
Achiral Nucleoside Analogue
Thomas Boesen, Daniel Sejer Pedersen, Jacob Jensen, Michael T. Munck,
Brian M. Nielsen, Asger B. Petersen, Ulla Henriksen, Britta M. Dahl,
*
and Otto Dahl
Department of Chemistry, University of Copenhagen, The H. C. Ørsted Institute,
Copenhagen, Denmark
ABSTRACT
Preparation of the nucleoside analogues 1 and incorporation of 1, B ¼ T, in deoxy-
ribooligonucleotides by the phosphoramidite method is described. A two-step
deprotection procedure was developed to reduce cleavage of the modified
allylic unit. The binding properties of the modified oligonucleotides towards
complementary DNA and RNA has been evaluated by Tm measurements
showing a DTm of ꢀ2 to ꢀ6.5ꢁC per modification. An oligonucleotide with
two modifications at the 30-end showed considerable resistance towards clea-
vage by a 30-exonuclease. No antiviral activity against HIV-1 or HSV-1 was
found for 1, B ¼ G or T, or for any of the trihydroxy derivatives 5.
Key Words: Nucleoside analogues; Modified oligonucleotides; Antiviral agents.
*Correspondence: Otto Dahl, Department of Chemistry, University of Copenhagen, The
H. C. Ørsted Institute, Universitetsparken 5, DK-2100 Copenhagen, Denmark; Fax: +45
35 52 02 12; E-mail: dahlo@kiku.dk.
623
DOI: 10.1081/NCN-120021967
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

624
Boesen et al.
INTRODUCTION
Since the invention of Acyclovir and PNA, the search has been intensified for
simple acyclic, preferable achiral nucleoside analogues as potential antisense mono-
mers or antiviral compounds. We have initiated a study of one such type of com-
pounds, 1, N-1=9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases, which are
acyclic, achiral analogues conformationally restricted by a C¼C bond.
Model studies indicated that the nucleobase and the two hydroxy groups of 1
can be positioned close to the positions of the nucleobase and the 30- and 50-OH
groups in ribonucleosides in the North conformation, as well as to the positions
of the nucleobase and the 30- and 50-OH groups in deoxyribonucleosides in the South
conformation. This is one of the prerequisites for good binding to RNA, resp. DNA.
PREPARATION
We have published the preparation of the thymine analogue,[1] and the A, G and
U analogues can be obtained in a similar way. Only the G analogue had been
described previously as a potential antiviral agent, without any biological data.[2]
Our improved synthesis of the T analogue is shown in Sch. 1. The C analogue has
to be prepared from the U analogue since in the last step of the procedure (Sch. 1),
removal of the benzyl groups with BCl3, the C analogue (1, B ¼ CBz) decomposed.
The thymine analogue has been built into DNA oligonucleotides by phosphor-
amidite chemistry. Dimethoxytritylation of 1, B ¼ T, gave a mixture of the two
mono-DMT derivatives 2a and 2b and the bis-DMT derivative 3 which were easily
Scheme 1.

Acyclic, Achiral Nucleoside Analogue
625
Scheme 2.
separated by column chromatography on silica. Standard phosphitylation of 2a and
2b gave the Ta and the Tb phosphoramidites 4a and 4b (Sch. 2).
The modified phosphoramidites were manually coupled with tetrazole activation
using a LCAA-CPG support. After an aqueous wash, capping, and oxidation with
ButOOH, the DMT efficiency was 99–100%. However, the next coupling following
a Tb unit proceeded with only 80–85% DMT-efficiency, irrespective of the type of
amidite or the coupling time. The reason for this is at present unknown. Deprotec-
tion and cleavage from the support was performed by a two-step procedure: 1) neat,
anhydrous diisopropylamine, 14 h at rt to remove the cyanoethyl groups;[3] 2) conc.
aq. ammonia, 2 h at rt. If aq. ammonia alone was used the modified T unit was
rapidly cleaved, probably by nucleophilic attack at the allylic position by NH3 or
OHꢀ. After removal of the cyanoethyl groups, the modified oligonucleotides were
totally stable in water at pH 7 and less than 1% cleaved by conc. aq. ammonia after
24 h at rt. Several oligonucleotides containing one Ta or one or two Tb units (Table)
were prepared, purified by ion exchange HPLC, and characterised by CE and
ES-MS.
PROPERTIES
The modified oligonucleotides hybridised to DNA and RNA, although with
reduced affinity (Table). The results for Tb, DTm ꢀ2 to ꢀ6.5ꢁC per modification,
was better than that for Ta, as expected. A mismatch of C opposite Tb gave a large
depression (DTm ꢀ15.5ꢁC) which indicates that the modified monomer discriminates
well against mismatches.
The resistance of the modified unit towards 30-exonucleolytic cleavage was eval-
uated on dT11TbTbT. Under conditions where a dT14 was cleaved by SVP with a t1=2

626
Boesen et al.
Table 1. Hybridization data (Tm,ꢁC) for modified and unmodified oligodeoxyribonucleotides
with DNA and RNA complements.a
b
dA14
DTm
rA14
DTm rA6CA7 DTm
dT14
36.0
31.0
26.0
31.0
33.5
18.0
12.5
ꢀ15.5
ꢀ15.5
dT7TbT6
dT7TaT6
dT11TbTbT
ꢀ5.0 28.0
ꢀ10.0 27.0
ꢀ2.5 29.0
ꢀ5.5
ꢀ6.5
ꢀ2.0
dGTGAGATGC DTm
rGTGAGATGC DTm
dGCATCTCAC
39.0
41.0
ꢀ5.5 27.5
dGCATbCTbCAC 28.0
ꢀ6.5
aTm was determined by measuring absorbance at 260 nm against increasing temperature
(0.5ꢁC steps) on equimolar mixtures (3 mM in each strand) of modified oligomer and its com-
plementary DNA or RNA strand in medium salt buffer (10 mM Na2HPO4, 100 mM NaCl,
0.1 mM EDTA, pH 7.0). Ta and Tb are explained in the text.
bChange in Tm per modification.
of 8 min, dT11TbTbT (apart from the first dT unit) was only ca. 35% cleaved after 2 h,
i.e., t1=2 > 3 h.
Antiviral activities against HIV-1 and HSV-1 of 1, B ¼ G, T, and the saturated
trihydroxy derivatives 5, were evaluated. None of the compounds showed any signi-
ficant effect at 30 or 300 mM.
CONCLUSION
The very simple acyclic, achiral nucleoside monomer 1, B ¼ T, is able to substi-
tute a DNA monomer in oligonucleotides, albeit the binding to DNA and RNA
complements is reduced. Work is in progress to optimise the coupling and deprotec-
tion procedures in order to prepare and study fully modified oligomers, and to
examine possible reasons (less than optimal preorganised conformation, reduced sol-
vation, or other factors) for the reduced binding properties. The hitherto examined
nucleoside analogues had no antiviral effects towards HIV-1 or HSV-1.
ACKNOWLEDGMENTS
We thank Dr. Claus Nielsen, Statens Serum Institute, Copenhagen, for the anti-
viral measurements, Cureon A=S for the ES-MS determinations, Ms. Jette Poulsen
and Ms. Anette W. Jørgensen for expert technical assistance, and the Danish Nat-
ural Science Research Council and The Danish Technical Research Council for
financial support.

Acyclic, Achiral Nucleoside Analogue
627
REFERENCES
1. Sejer Pedersen, D.; Boesen, T.; Eldrup, A.B.; Kiær, B.; Madsen, C.; Henriksen,
U.; Dahl, O. The first example of a new type of acyclic, achiral nucleoside
analogue: 1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]thymine. J. Chem. Soc.,
Perkin Trans. 2001, 1, 1656.
2. Nakayama, T.; Ito, T.; Morisawa, Y.; Ikeuchi, K.; Shinko, S.; Takase, H.;
Murakami, Y. Preparation of acyclic purine nucleoside derivatives as antiviral
agents, JP7048374, February 22, 1995, in Japanese.
3. Alul, R.H.; Singman, C.N.; Zhang, G.; Letsinger, R.L. Nucleic Acids Res. 1991,
19, 1527.