N2′-functionalized 2′-amino-α-L-LNA adenine derivatives - Efficient targeting of single stranded DNA

Article abstract of DOI:10.1080/15257770701539153

The synthesis of two pyrene-functionalized 2α-amino-α-L-LNA adenine building blocks is outlined and initial results from thermal denaturation studies are presented. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701539153

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N2′-Functionalized 2′-Amino-α-L-LNA
Adenine Derivatives—Efficient Targeting
of Single Stranded DNA
Nicolai K. Andersen a , Jesper Wengel a & Patrick J. Hrdlicka a b
a Nucleic Acid Center, Department of Physics and Chemistry,
University of Southern Denmark, Odense M, Denmark
b Department of Chemistry, University of Idaho, Moscow, Idaho, USA
Available online: 05 Dec 2007
To cite this article: Nicolai K. Andersen, Jesper Wengel & Patrick J. Hrdlicka (2007): N2′-
Functionalized 2′-Amino-α-L-LNA Adenine Derivatives—Efficient Targeting of Single Stranded DNA,
Nucleosides, Nucleotides and Nucleic Acids, 26:10-12, 1415-1417
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1415–1417, 2007
Copyright ꢀ Taylor & Francis Group, LLC
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ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701539153
N2ꢀ-FUNCTIONALIZED 2ꢀ-AMINO-α-L-LNA ADENINE
DERIVATIVES—EFFICIENT TARGETING OF SINGLE STRANDED DNA
Nicolai K. Andersen and Jesper Wengel
Nucleic Acid Center, Department
2
of Physics and Chemistry, University of Southern Denmark, Odense M, Denmark
Patrick J. Hrdlicka Nucleic Acid Center, Department of Physics and Chemistry,
2
University of Southern Denmark, Odense M, Denmark; Department of Chemistry, University
of Idaho, Moscow, Idaho, USA
The synthesis of two pyrene-functionalized 2ꢁ-amino-α-L-LNA adenine building blocks is out-
2
lined and initial results from thermal denaturation studies are presented.
Keywords Conformationally restricted nucleosides; antisense; antigene
INTRODUCTION
Motivated by the potential of LNA[1] and α-L-LNA[2] within nucleic acid
based therapeutics and diagnostics, we have recently explored the corre-
sponding 2ꢁ-amino analogs, which allow extensive chemical derivatization
of nucleic acids.[3–5] Incorporation of pyrene-functionalized 2ꢁ-amino-α-L-
LNA thymine monomers into short DNA strands, results in dramatically in-
creased thermal affinity towards DNA complements relative to unmodified
reference strands.[4] This property has been used to develop a novel strategy
for targeting of double-stranded DNA.[4] Due to the challenging synthetic
route towards these building blocks,[5] we set out to synthesize the corre-
sponding 2ꢁ-amino-α-L-LNA adenine derivatives described herein.
RESULTS AND DISCUSSION
The developed synthetic route to the desired pyrene-functionalized
2ꢁ-amino-α-L-LNA adenine phosphoramidites 10 and 12 initiates from the
The Danish National Research Foundation is thanked for financial support.
Address correspondence to Patrick J. Hrdlicka, Department of Chemistry, University of Idaho,
Moscow, ID 83843-2343. E-mail: Hrdlicka@uidaho.edu
1415

1416
N. K. Andersen et al.
SCHEME 1 Reagents and conditions: a) (CF3SO2)2O, an. pyridine, an. CH2Cl2, −78◦C; b) NaN3, 15-
crown-5, an. DMF, 40◦C, 89% (over 2 steps); c) 2 M aq. NaOH, PMe3, THF, rt, 85%; d) (CF3CO)2O, an.
pyridine, an. CH2Cl2, 0◦C, 62%; e) BCl3, an. CH2Cl2, −78◦C to rt, 87%; f) NaOBz, 15-crown-5, an. DMF,
90◦C, 83%; g) 2 M aq. NaOH, 1,4-dioxane/H2O, 0◦C, 60%; h) DMTrCl, an. pyridine, 0◦C to rt, 34%;
i) PyCHO, NaBH(OAc)3, 1,2-DCE, rt, 68%; j) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, 1,2-DCE, rt, 51%; k)
PyCOOH, EDC·HCl, CH2Cl2, rt, 64%; l) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, 1,2-DCE, rt, 67%; m) DNA
synthesizer. ABz: 6N-benzoyladenine-9-yl; Py: pyren-1-yl.
known adenine derivative 1 (Scheme 1).[2] O2ꢁ-Triflation of alcohol 1 fol-
lowed by nucleophilic substitution affords azido nucleoside 2, which upon
subjection to conditions favoring a tandem Staudinger/intramolecular
nucleophilic substitutions furnishes known bicyclic nucleoside 3[6] in
excellent yield. After a subsequent series of protecting group manip-
ulations, amino alcohol 8 was obtained, which upon chemoselective
N2ꢁ-functionalization via reductive amination or by EDC mediated coupling
afforded nucleosides 9 and 11, respectively. After standard phosphitylation
the desired amidites 10 and 12 was obtained as suitable building blocks for
incorporation into oligodeoxyribonucleotides (ONs) via automated DNA
synthesis.
ONs containing a single incorporation of monomer X or Y, exhibit
greatly increased thermal affinities towards complementary DNA (up to
+11.0◦C) while lower increases are observed toward RNA complements
(Table 1). Single nucleotide mismatches opposite of the site of incorpora-
tion are discriminated well (footnote Table 1). Full details of this study will
be presented elsewhere.

N2ꢁ-Functionalized 2ꢁ-Amino-α-L-LNA Adenine Derivatives
1417
TABLE 1 Thermal denaturation temperatures (Tm values) of pyrene-functionalized
2ꢁ-amino-α-L-LNA towards DNA/RNA complementsa
Tm (ꢀTm)/◦C
DNA
RNA
ON1
ON2
ON3
5ꢁ-GCA TXT ACG
5ꢁ-GCA TYT ACG
5ꢁ-GCA TAT ACG
34.0 (+6.0)
39.0 (+11.0)
28.0
24.0 (−0.5)
27.0 (+2.5)
24.5
aTm values/◦C (ꢀTm = change in Tm value calculated relative to DNA:DNA or
DNA:RNA reference duplex) recorded in medium salt buffer ([Na+] = 110 mM,
[Cl−] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 µM concentrations of
each strand. Tm values (◦C) of ON1 and ON2 towards DNA strands containing a
single mismatch in the central position A/C/G: 18.5/27.0/19.5 and 18.0/27.0/22.0,
respectively. Tm values (◦C) of ON1 and ON2 towards RNA strands containing a sin-
gle mismatch in the central position A/C/G: 13.5/16.0/14.0 and 16.5/16.0/13.5,
respectively.
REFERENCES
1. Petersen, M.; Wengel, J. LNA: A versatile tool for therapeutics and genomics. Trends Biotechnol. 2003,
21, 74–81.
2. Sørensen, M.D.; Kværnø, L.; Bryld, T.; Ha˚kansson, A.E.; Verbeure, B.; Gaubert, G.; Herdewijn, P.;
Wengel, J. α-L-ribo-Configured locked nucleic acid (α-L-LNA): Synthesis and properties. J. Am. Chem.
Soc. 2002, 124, 2164–2176.
3. Hrdlicka, P.J.; Babu, B.R.; Sørensen, M.D.; Harrit, N.; Wengel, J. Multilabeled pyrene-functionalized
2ꢁ-amino-LNA probes for nucleic acid detection in homogenous fluorescence assays. J. Am. Chem. Soc.
2005, 127, 13293–13299 and references cited herein.
4. Hrdlicka, P.J.; Kumar, T.S.; Wengel, J. Targeting of mixed sequence double-stranded DNA using
pyrene-functionalized 2ꢁ-amino-α-L-LNA. Chem. Comm. 4279–4281.
5. Kumar, T.S.; Madsen, A.S.; Wengel, J.; Hrdlicka, P.J. Synthesis and hybridization studies of 2ꢁ-amino-
α-L-LNA and a tetracyclic ‘locked LNA.’ J. Org. Chem. 2006, 71, 4188–4201.
6. Sørensen, M.D.; Wengel, J.; Koch, T., Christensen, S.M.; Rosenbohm, C.; Pedersen, D.S. Synthesis of
locked nucleic acid derivatives. Patent application WO03095467.