Synthesis and biophysical studies of N2′-functionalized 2′-amino-α-L-LNA

Article abstract of DOI:10.1080/15257770701538841

A synthetic route towards a selected set of N-acylated and N-alkylated derivatives of 2′-amino-α-L-LNA phosphoramidite building blocks has been developed. Biophysical studies suggest that the 2-oxo-5-azabicyclo[2.2.1] heptane skeleton of 2′-amino-α-L-LNA

Full text of DOI:10.1080/15257770701538841

Nucleosides, Nucleotides, and Nucleic Acids, 26:1403–1405, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701538841
SYNTHESIS AND BIOPHYSICAL STUDIES OF N2ꢀ-FUNCTIONALIZED
2ꢀ-AMINO-α-L-LNA
T. Santhosh Kumar, Andreas S. Madsen, and Jesper Wengel
Nucleic Acid
2
Center, Department of Physics and Chemistry, University of Southern Denmark, Odense M,
Denmark
Patrick J. Hrdlicka
Department of Chemistry, University of Idaho, Moscow,
2
Idaho, USA
A synthetic route towards a selected set of N-acylated and N-alkylated derivatives of 2 ꢁ-amino-
2
α-L-LNA phosphoramidite building blocks has been developed. Biophysical studies suggest that the
2-oxo-5-azabicyclo[2.2.1]heptane skeleton of 2 ꢁ-amino-α-L-LNA allows precise positioning of inter-
calators in the core of nucleic acid duplexes.
Keywords 2ꢁ-Amino-α-L-LNA; pyrene; oligonucleotides; intercalation
INTRODUCTION
The high affinity hybridization of LNA,[1] 2ꢁ-amino-LNA,[2] α-L-LNA,[3]
and 2ꢁ-α-L-amino-LNA[4] toward complementary DNA/RNA complements
are well established. As an extension of our recent efforts to use N2ꢁ-
functionalized 2ꢁ-amino-LNA monomers as building blocks in nucleic acid
based diagnostics and therapeutics,[5] we have developed an interest in
N2ꢁ-functionalized 2ꢁ-amino-α-L-LNA building blocks. Among these, double
stranded 2ꢁ-N-(pyren-1-yl)methyl-2ꢁ-amino-α-L-LNA have been shown to tar-
get double-stranded DNA.[6] Herein, we present the synthesis and biophysi-
cal studies of N2ꢁ-functionalized 2ꢁ-amino-α-L-LNA.
RESULTS AND DISCUSSION
The synthesis of a selected set of N2ꢁ-functionalized 2ꢁ-amino-α-L-
LNA phosphoramidites is conveniently achieved in two steps from key
We greatly appreciate funding from The Danish National Research Foundation.
Address correspondence to T. Santhosh Kumar, Nucleic Acid Center, Department of Physics and
Chemistry, University of Southern Denmark, 5230 Odense M, Denmark. E-mail: skt@chem.sdu.dk
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T. S. Kumar et al.
SCHEME 1 Reagents and conditions: a) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, 56% for 3Q, 63% for
3S, 90% for 3V, 88% for 3W, 60% for 3X, 75% for 3Y and 36% for 3Z; b) DNA synthesizer; DMTr =
4,4ꢁ-dimethoxytrityl, Fmoc = 9ꢁ-fluorenylmethoxycarbonyl, Py = pyren-1-yl.
intermediate 1 by chemoselective carbamoylation (monomer Q) or EDC-
mediated N-acylation (monomers X-Z), reductive amination (monomers
S and W), or peracylation followed by selective deacylation (monomer
V, Scheme 1). Further details on the synthesis and incorporation of
N2ꢁ-functionalized 2ꢁ-amino-α-L-LNA phosphoramidites 3Q-Z into short
oligodeoxyribonucleotides (ONs) will be presented elsewhere.
Incorporation of a single pyrene functionalized 2ꢁ-amino-α-L-LNA
monomer (W-Z) results in dramatic increases in duplex stability with
DNA complements of up to +19.5◦C and significantly smaller increases
in duplex stability with RNA complements. A single incorporation of
non-functionalized 2ꢁ-amino-α-L-LNA monomer Q results in comparably
more modest increases in duplex stability with DNA/RNA complements
(Table 1).[4] Surprisingly, single incorporations of ethyl or acetyl substi-
tuted 2ꢁ-amino-α-L-LNA monomers (S or V) into ONs result in greatly
decreased thermal affinities towards its DNA/RNA complements. The ob-
served DNA selectivity (Table 1), limited mismatch discrimination, molec-
ular modeling studies and hybridization induced bathocromic shifts of
pyrene absorption maxima (data not shown), suggest that the 2-oxo-5-
azabicyclo[2.2.1]heptane skeleton of these monomers positions the pyrene
moiety suitably for intercalation upon hybridization.

N2 ꢁ-Functionalized 2 ꢁ-Amino-α-L-LNA
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TABLE 1 Thermal denaturation temperatures of duplexes formed by 5ꢁ-d(GCAB AT CAC)a
and DNA/RNA complementsb
[Tm (ꢀTm/mod)/◦C]
B = Q
B = S
B = V
B = W
B = X
B = Y
B = Z
DNA 31.0 (+2.5) 22.5 (−6.0) 19.0 (−9.5) 44.0 (+15.5) 48.0 (+19.5) 45.0 (+16.5) 35.0 (+6.5)
RNA 29.0 (+4.5) 21.5 (−3.0) 20.5 (−4.0) 32.0 (+7.5) 36.0 (+11.5) 36.5 (+12.0) 31.0 (+6.5)
aTm values of unmodified duplex (where B = T) toward its complementary DNA and RNA are 28.5◦C
and 24.5◦C, respectively.
b Thermal denaturation temperatures recorded in medium salt buffer ([Na+] = 110 mM, [Cl−] = 100
mM, pH 7.0 (adjusted with 10 mM NaH2PO4/5 mM Na2HPO4)), using 1.0 µM concentrations of the
two complementary strands. See synthetic scheme for structure of monomers.
REFERENCES
1. Wengel, J. Synthesis of 3ꢁ-C- and 4ꢁ-C-branched oligodeoxynucleotides and the development of locked
nucleic acid (LNA). Accounts Chem. Res. 1999, 32, 301–310.
2. Singh, S.K.; Kumar, R.; Wengel, J. Synthesis of 2ꢁ-amino-LNA: A novel conformationally restricted
high-affinity oligonucleotide analogue with a handle. J. Org. Chem. 1998, 63, 10035–10039.
3. 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.
4. Kumar, T.S.; Madsen, A.S.; Wengel, J.; Hrdlicka, P.J. Synthesis and hybridization studies of 2ꢁ-amino-
α-L-LNA and tetracyclic “Locked LNA.” J. Org. Chem. 2006, 71, 4188–4201.
5. 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 homogeneous fluorescence assays. J. Am. Chem.
Soc. 2005, 127, 13293–13299 and references cited therein.
6. 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. 2005, 4279−4281.