Attachment of cholesterol to amino-LNA: Synthesis and hybridization properties

Article abstract of DOI:10.1080/15257770701549095

Here, we present our synthesis of amino-LNA with a C6-linker and hybridization studies of these. A cholesterol moiety was attached at the end of the C6-linker. This resulted in drastic drops against DNA of the modified oligonucleotide. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701549095

Nucleosides, Nucleotides, and Nucleic Acids, 26:1645–1647, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701549095
ATTACHMENT OF CHOLESTEROL TO AMINO-LNA:
SYNTHESIS AND HYBRIDIZATION PROPERTIES
Torsten Bryld and Christian Lomholt
Exiqon A/S, Vedbæk, Denmark
2
Here, we present our synthesis of amino-LNA with a C6-linker and hybridization studies of
2
these. A cholesterol moiety was attached at the end of the C6-linker. This resulted in drastic drops
against DNA of the modified oligonucleotide.
Keywords LNA; amino-LNA; cholesterol
Oligonucleotides modified with LNA[1] monomers (Figure 1) have
demonstrated an unprecedented high affinity toward complementary DNA
and RNA with increases in the melting temperature (Tm) up to 10◦C per
modification. The amino-LNA (Figure 1) monomers have demonstrated
similar hybridization properties.[2] The secondary amino group of amino-
LNA can be regarded as a handle for the attachment of various groups.
The attachment of cholesterol to miRNA knockdown probes has resulted
in increased activity of those.[3] We wanted to explore the opportunity of
introducing several cholesterol units to knockdown probes by utilizing the
handle of amino-LNA. In order to have the effect of the cholesterol unit this
was introduced to the amino-LNA via a C6 linker (Figure 1).
The known nucleoside 1[2] (Scheme 1) was alkylated with phtal-
imidohexanal[4] in the presence of NaCNBH3 to nucleoside 2 in 51%
yield. Nucleoside 3 was obtained by protection of the primary hydroxy
group with a DMT group using DMTCl in pyridine in 56% yield. Subse-
quently the phtalimide group was removed by treatment with hydrazine
affording nucleoside 4 in 70% yield having a primary amino group ready
for functionalization. The cholesterol group was introduced by formation
of amide 5 in 53% yield by a chemoselective reaction with cholesteryl
chloroformate in the presence of pyridine. Amide 5 was transformed
into phosphoramidite 6 using standard conditions in a yield of 42%.
Key intermediate 4 was also transformed into nucleoside 7 using ethyl
Address correspondence to Torsten Bryld, Exiqon A/S, Bygstubben 9, 2945 Vedbæk, Denmark.
E-mail: tbr@exiqon.com
1645

1646
T. Bryld and C. Lomholt
FIGURE 1 LNA and analogs.
trifluoroaceate. This was subsequently transformed into phosphoramidite 8
using the same conditions as for phosphoramidite 6. Phosphoramidite 6 and
8 gave monomers X and Y when incorporated in oligonucleotides vide infra.
The synthesis of monomers X and Y was achieved. The hybridization
studies of ONs modified with monomer Y show that this modification
induced an increased towards complementary DNA resulting in ꢀTm/mod.
between 1 and 9◦C (Table 1). These results are similar to those of amino-
LNA. Introduction of the cholesterol group did, however, resulted in
dramatic decreases in Tm of the modified ONs when hybridized toward
SCHEME 1 i) 6-Phtalimidohexanal, NaCNBH3, MeOH; ii) DMTCl, pyridine; iii) H2NNH2, EtOH, pyri-
dine, acetic acid; iv) Cholesteryl chloroformate, CH2Cl2, pyridine; v) ((iPr)2N)2PO(CH2)2CN, DCI,
CH2Cl2 vi) CF3COOEt, Et3N; vii) ((iPr)2N)2PO(CH2)2CN, DCI, CH2Cl2.

Cholesterol Attachment to Amino-LNA
1647
TABLE 1 Thermal denaturation temperatures measured as the maximum of the first derivative of the
melting curve (A260 versus temperature; 5◦C to 80◦C with an increase of 1◦C/minute) recorded in
medium salt buffer (100 mM NaCl, 10 mMNaH2PO4 0.2 mM EDTA, pH 7.0)
5ꢁ-d(GATAGCGAAGA)
Tm ◦C
ꢀTm/mod. ◦C
5ꢁ-d(TCTTCGCTATC)
5ꢁ-d(TCTTCGCTAXC)
5ꢁ-d(TCTTCGCTAYC)
5ꢁ-d(XCTTCGCTATC)
5ꢁ-d(YCTTCGCTATC)
5ꢁ-d(TCXTCGCTATC)
5ꢁ-d(TCYTCGCTATC)
5ꢁ-d(TCTTCGCXATC)
5ꢁ-d(TCTTCGCYATC)
5ꢁ-d(TCTXCGCXATC)
5ꢁ-d(TCTYCGCYATC)
5ꢁ-d(XCTXCGCXATC)
5ꢁ-d(YCTYCGCYATC)
5ꢁ-d(XCTTCGCTAXC)
5ꢁ-d(YCTTCGCTAYC)
5ꢁ-d(XCTXCGCTATC)
5ꢁ-d(YCTYCGCTATC)
34.2
32.6
38.5
30.2
35.4
30.2
37.6
32.2
43.1
<10
44.8
<10
44.8
<10
38.6
<10
36.1
ref.
−1.6
+4,3
−4.0
+1.2
−4.0
+3.4
−2.0
+8.9
>−12.1
+5.3
>−8.1
+3.5
>−12,1
+2.2
>−12,1
+1.0
complementary DNA. The incorporation of more than 2 X monomers in
an 11-mer led to Tm’s lower than 10◦C. This effect can be contributed to a
steric effect of the cholesterol groups, having the cholesterol groups inter-
fering with the nucleobases. We, therefore, conclude that this construct was
unsuited for the use in knock-down probes.
REFERENCES
1. Koshkin, A.A.; Singh, S.K.; Nielsen, P.; Rajwanshi, V.K.; Kumar, R.; Meldgaard, M.; Olsen, C.E.;
Wengel, J. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine,5-
methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprece-
dented nucleic acid recognition. Tetrahedron 1998, 3607–3630.
2. Sorensen, M.D.; Petersen, M.; Wengel, J. Functionalized LNA (locked nucleic acid): high-affinity
hybridization of oligonucleotides containing N -acylated and N -alkylated 2ꢁ-amino-LNA monomers.
Chem. Commun. 2003, 2130–2131.
3. Kru¨tzfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K.G.; Tuschl, T.; Manoharan, M.; Stoffel, M. Silencing
of microRNAs in vivo with ‘antagomirs.’ Nature 2005, 438, 685–689.
4. Agathocleous, D.C.; Page, P.C.B.; Cosstick, R.; Galpin, I.J.; McLennan, A.G.; Prescott, M. Synthesis
of bridged dinucleosides. Tetrahedron 1990, 46 (6), 2047–2058.