Photochemically induced RNA and DNA abasic sites

Article abstract of DOI:10.1080/15257770701527711

Two phosphoramidite building blocks were synthesized that can easily be deprotected by UV light to reveal natural abasic sites in oligoribonucleotides as well as in oligodeoxyribonucleotides. Another building block which releases a 2′-O-methylated abasic

Full text of DOI:10.1080/15257770701527711

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Nucleosides, Nucleotides and Nucleic
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Photochemically Induced RNA and DNA
Abasic Sites
Pascal A. Küpfer a & Christian J. Leumann a
a Department of Chemistry and Biochemistry , University of Bern ,
Bern, Switzerland
Published online: 10 Dec 2007.
To cite this article: Pascal A. Küpfer & Christian J. Leumann (2007) Photochemically Induced
RNA and DNA Abasic Sites, Nucleosides, Nucleotides and Nucleic Acids, 26:8-9, 1177-1180, DOI:
10.1080/15257770701527711
To link to this article: http://dx.doi.org/10.1080/15257770701527711
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1177–1180, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701527711
PHOTOCHEMICALLY INDUCED RNA AND DNA ABASIC SITES
Pascal A. Ku¨ pfer and Christian J. Leumann
Department of Chemistry and
2
Biochemistry, University of Bern, Bern, Switzerland
Two phosphoramidite building blocks were synthesized that can easily be deprotected by UV
2
light to reveal natural abasic sites in oligoribonucleotides as well as in oligodeoxyribonucleotides.
Another building block which releases a 2 ꢁ-O-methylated abasic site upon UV radiation is also
described.
Keywords RNA; DNA; natural abasic sites; UV radiation
The design of potential assays using true abasic site containing oligonu-
cleotides demands the final deprotection step to release the potentially un-
stable abasic lesion to occur fast, under mild conditions and merely quanti-
tative in order not to interfere unnecessarily with the assay conditions. More-
over the deprotection conditions of a chosen protecting group have to be
orthogonal to standard oligonucleotide chemistry to ensure a wide usability
of the designed phosphoramidite building blocks.
The 2-nitrobenzyl moiety (NB) has been used for the protection of the
1ꢁ-O-position of a deoxyribose unit before.[1] The NB unit, also used to cage
the 2ꢁ-hydroxy position of the furanose ring, has shown not to be fully stable
when treated with fluoride ions used in standard RNA deprotection of 2ꢁ-
O-TBDMS groups.[2] Clearly, a 2-nitrobenzyl derivative with higher stability
against fluoride treatment was needed.
The 1-(2-nitrophenyl)ethyl (1NPE) unit was shown to be fully stable un-
der the deprotection conditions mentioned above. Besides this, deprotec-
tion of the 1NPE unit gives rise to the less reactive 2-nitrosoacetophenone
compared to the biologically toxic 2-nitrosobenzaldehyde that arises from
2-nitrobenzyl deprotection. 1NPE adducts are additionally known to pho-
tolyse 20 times more rapidly than the NB adducts.[3]
The synthesis of the RNA abasic site precursor (Scheme 1) started
with the formation of the acetal 5 under Vorbru¨ggen conditions using
Address correspondence to Christian J. Leumann, Department of Chemistry and Biochemistry,
University of Bern, Freiestrasse 3, 3012 Bern, Switzerland; E-mail: leumann@ioc.unibe.ch
1177

1178
P. A. Ku¨pfer and C. J. Leumann
SCHEME 1 a) Tetra-O-acetyl-ribofuranose (1.2 eq.), TMSOTf (0.35 eq.) added in 4 portions over 90
minutes, CH3CN, 2 hours, −20◦C, 77%. b) Na2CO3 (1 eq.), MeOH, 22 hours, r.t., 90%. c) DMT-C1 (1.2
i
eq.), pyridine, 1.5 hours, r.t., 94%. d) 1) Bu2SnCl2 (1.1 eq.), Pr2NEt (3.5 eq.), (CH2Cl)2, 1 hour, r.t.,
2) (8) (1.1 eq.), 30 minutes, 80◦C, 94% for both isomers. e) [(i Pr2N)(NCCH2CH2O)P]Cl (1.45 eq.),
i Pr2NEt (2.9 eq.), THF, 14 hours, r.t., 87%.
SCHEME 2 a) TIPDS-Cl (1:2 eq.), pyridine, 18 hours, r.t., 82%. b) AgO (8 eq.), MeI, 72 hours, 50◦C,
90%. c) TBAF (3 eq.), acetic acid (1.6 eq.), THF, 30 minutes, r.t., 85%. d) DMT-Cl (1.2 eq.), pyridine, 1.5
hours, r.t., 94%. e) [(i Pr2N)(NCCH2CH2O)P]Cl (1.45 eq.), i Pr2NEt (2.9 eq.), THF, 4 hours, r.t., 78%.

RNA and DNA Abasic Sites
1179
SCHEME 3 a) Tri-O-acetyl-2ꢁ-deoxyribofuranose (0.7 eq.), TMSOTf (0.2 eq.) added dropwise, CH3CN,
1 hour, −20◦C, 84%. b) Na2CO3 (1 eq.), MeOH, 22 hours, r.t., 91%. c) DMT-Cl (1.2 eq.), pyridine, 1.5
hours, r.t., 74%. d) [(i Pr2N)(NCCH2CH2O)P]Cl (1.45 eq.), i Pr2NEt (2.9 eq.), THF, 1 hour, r.t., 77%.
enantiopure 1-(R)-(2-nitrophenyl)ethanol 4a, synthesized after the method
of Corrie et al.,[4] and commercially available (1,2,3,5)-tetra-O-acetyl-
ribofuranose. The pure β-anomer 5 was deacetylated and DMT-protected
by standard methods to give nucleoside 7. Preparation of [(triisopropy-
lsilyl)oxy]methyl) chloride 8 and subsequent alkylation of the 2ꢁ-hydroxy
group of the ribose was performed in analogy to known methods.[5] The
ratio of the 2ꢁ- and 3ꢁ- regioisomers 9a and 9b was determined to be 65% in
favor of the 2ꢁ-O-TOM protected nucleoside 9a, according to 1H-NMR anal-
ysis. Repeated column chromatography yielded 37% of the pure 2ꢁ-O-TOM
nucleoside and 57% of an inseparable 2ꢁ-, 3ꢁ-O-TOM mixture. Reaction of 9a
with 2-cyanoethyl diisopropylchlorophosphoramidite finally gave the abasic
RNA building block 1.
Synthesis of the 2ꢁ-O-Me RNA abasic site precursor (Scheme 2) started
with intermediate 6 that was TIPDS protected to give 10. The 2ꢁ-hydroxyl
group was then methylated followed by deprotection of the TIPDS group
with TBAF to yield diol 12. Protection with DMT-chloride and phosphityla-
tion finally gave the 2ꢁ-OMe-abasic building block 2.
If for both synthetic pathways a mixture of diastereomers of 6, arising
from the use of an enantiomeric mixture of 1(2-nitrophenyl)ethanol is used,
the two isomers can be separated by column chromatography at the stage
of intermediate 7 or 10, respectively. Enantiopure 1-(2-nitrophenyl)ethanol
as starting material is therefore not needed in order to get diastereopure
products.
The synthesis of the DNA abasic site precursor (Scheme 3) started with
(1ꢁ,3ꢁ,5ꢁ)-Tri-O-acetyl-2ꢁ-deoxy-D-ribofuranose,[6] which was transformed into
acetal 14 with enantiopure 1-(S)-(2-nitrophenyl)ethanol 4b as described
for the RNA abasic site precursor with comparable yields. The remain-
ing steps were performed with the mixture of anomers although at the
stages of the intermediates 14 and 15 the anomers were separable accord-
ing to TLC. Phosphoramidites 1–3 were incorporated in oligonucleotides
using standard nucleic acid synthesis procedures. The modified phospho-
ramidites were allowed to couple for 6 minutes and 12 minutes for DNA

1180
P. A. Ku¨pfer and C. J. Leumann
FIGURE 1 Heptamers 5ꢁ-AGG-1-UUC-3ꢁ ( ) and 5ꢁ-AGG-3-TTC-3ꢁ ( ) are deprotected with a UV lamp
ꢀ
◦
(150 W). Deprotection kinetics show half-life times of 12.5 seconds and 3.4 seconds for the deprotection
of RNA and DNA abasic sites, respectively. Logarithmic plots prove the first order character of the re-
actions and give the rate constants k = 5.68 · 10−2s−1 for RNA and k = 2.11 · 10−1s−1 for DNA for the
deprotection reaction.
and RNA, respectively. 2ꢁ-O-TOM groups were removed using the normal
silyl deprotection procedure of oligoribonucleotides.
In order to measure deprotection kinetics, phosphoramidite building
blocks 1 and 3 were incorporated into the heptamers 5ꢁ-AGG-1-UUC-3ꢁ and
5ꢁAGG-3-TTC-3ꢁ. Aqueous solutions of the oligonucleotides at a concentra-
tion of 1 OD260mL−1 in a quartz cuvette were irradiated with a UV lamp
(medium-pressure mercury vapor lamp, 150 W). The deprotection was fol-
lowed by RP-HPLC. From the exponential fits, half-life times of 12.5 seconds
(k = 5.68 · 10−2s−1) and 3.4 seconds (k = 2.11 · 10−1s−1) for the deprotec-
tion of RNA and DNA abasic site were determined. The collected data show
the deprotection reactions to be of first order kinetics. Oligonucleotides
containing building blocks 1–3 were also deprotected in almost quantitative
yield by exposition to the glass-filtered light of a slide projector (tungsten
lamp, 250 W) for 6 minutes. Especially the latter method shows a very mild
way to produce abasic sites from the presented building blocks with a very
low risk of inducing photolesions in oligonucleotides.
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3. Walker, J.W.; McCray, J.A.; Hess, G.P. Biochemistry 1986, 25, 1799–1805.
4. Corrie, J.E.T.; Reid, G.P.; Trentham, D.R.; Hursthouse, M.B.; Mazid, M.A. J. Chem. Soc., Perkin Trans.
1 1992, 1015–1019.
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