Synthesis and properties of oligonucleotides containing a cholesterol thymidine monomer

Article abstract of DOI:10.1080/15257770701501534

Highly selective base-pair recognition makes DNA a suitable building block for orderly self-assembled structures. For some applications in nanotechnology DNA complexes need to be fixed onto surfaces. To fulfil this requirement on lipid membranes we have synthesised a thymidine monomer modified with a cholesterol moiety. Solution studies show that the melting temperature (Tm) of the duplex, with adjacent cholesterols on each strand, is much higher than that of the unmodified duplex. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701501534

Nucleosides, Nucleotides, and Nucleic Acids, 26:785–794, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701501534
SYNTHESIS AND PROPERTIES OF OLIGONUCLEOTIDES
CONTAINING A CHOLESTEROL THYMIDINE MONOMER
Adeline Durand and Tom Brown
2
School of Chemistry, University of Southampton,
Highfield, Southampton, UK
2
Highly selective base-pair recognition makes DNA a suitable building block for orderly self-
assembled structures. For some applications in nanotechnology DNA complexes need to be fixed onto
surfaces. To fulfil this requirement on lipid membranes we have synthesised a thymidine monomer
modified with a cholesterol moiety. Solution studies show that the melting temperature (T ) of the
m
duplex, with adjacent cholesterols on each strand, is much higher than that of the unmodified
duplex.
Keywords Cholesterol; nonotechnology; DNA; fluorescence melting; self-assembly
INTRODUCTION
Double helical DNA is an attractive molecule for the design of nano-
[
1−3]
networks due to its inherent rigidity and highly selective base pairing.
Indeed, complexes formed by simple oligonucleotides (ODNs) have been
shown to self-assemble into orderly double-stranded structures on solid
[
4−7]
surfaces.
In this context we have an interest in assembling ODN ar-
[8]
rays on soft lipid membranes. To this end, we have synthesized a choles-
terol phosphoramidite monomer, which can be incorporated into DNA se-
quences at thymidine sites. ODNs modified with cholesterol have previously
[
9−13]
been synthesized for various purposes.
For example Hook et al. used a
ꢁ
ꢁ
cholesterol, attached at either the 3 - or 5 -end of the ODN, to tether them
to supported lipid bilayers.[
14,15]
For our purposes it is important to be able
to pin the nano-network to the lipid surface at any desired locus. Therefore,
we designed a phosphoramidite monomer, Chol-dT (Figure 1, A), which
can be positioned at any thymidine site within the synthetic ODN, without
disrupting base pairing (Figure 1, B). The monomer is functionalized with
This work was funded by the European Commission’s 6th Framework Programme and oligonu-
cleotide synthesis was carried out by ATDBio Ltd.
Address correspondence to Tom Brown, School of Chemistry, University of Southampton, High-
field, Southampton SO17 1BJ, UK. E-mail: tb2@soton.ac.uk
7
85

7
86
A. Durand and T. Brown
FIGURE 1 (A) Structure of the phosphoramidite monomer Chol-T. (B) Duplex anchored to a lipid
bilayer via cholesterol.
SCHEME 1 (i) a) Ethyl trifluoroacetate (1.5 eq), TEA (3 eq), MeOH, 22.5 h, rt; b) propargylamine (1
eq), EDC (1.3 eq), DCM, 24 h, rt, 79% overall; (ii) Ac2O (3 eq), pyridine, rt, 16.5 h, quant.; (iii) a)
◦
Ethanolic methylamine (70 eq), 55 C, 20 h; b) deprotected amine (2.2 eq), CuI (0.4 eq), (Ph3P)4Pd
(
0.2 eq), TEA (40 eq), DMF, rt, 30 h, dark, 73% overall; (iv) Cholesteryl chloroformate (1.2 eq), DIPEA
◦
(1.2 eq), DCM and DMF, 2 h at 0 C and 2 h at rt, 32%; (v) a) K2CO3 0.4M in MeOH:H2O 4:1(6 eq),
ꢁ
1
.75 h, rt; b) 4,4 -dimethoxytritylchloride (3 eq), pyridine, DCM, rt, 4.5 h, 45% overall; (vi) 2-cyanoethyl-
N ,N -diisopropyl chlorophosphine (1.1 eq), DIPEA (2.5 eq), THF, rt, 0.5 h, 82%.

Oligonucleotides Containing Cholesterol Thymidine Monomer
787
cholesterol and a hydrophobic spacer unit, which forms a bridge to the po-
lar anionic phosphate backbone of the DNA chain.
RESULTS AND DISCUSSIONS
The synthetic route to the Chol-dT monomer is outlined in Scheme 1.
Firstly, the commercially available 12-aminododecanoic acid was protected
with trifluoroacetyl and coupled with propargylamine to give the spacer 1
ꢁ
in 79% overall yield. 5-iodo-2 -deoxyuridine was acetylated to give 3 quan-
titatively and spacer 1 was deprotected and coupled via a Sonogashira re-
action to give the protected thymidine 4. The free amino group of com-
pound 4 was then coupled with cholesteryl chloroformate in 32% yield.
The acetyl protection groups were removed by treating with potassium car-
bonate in a mixture of methanol and water and dimethoxytritylation of the
resultant free nucleoside afforded 6, which was converted to its 2-cyanoethyl-
N ,N -diisopropyl phosphoramidite 7 in 82% yield. Monomer 7 was incorpo-
ꢁ
ꢁ
rated into the 18-mer ODNs 8 [5 -ACCAAGGTGCTATCGTCG-3 , T = Chol-
ꢁ
ꢁ
dT] and its complementary strand 9 [5 -CGACGATAGCACCTTGGT-3 , T =
Chol-dT] by automated solid-phase ODN synthesis. All ODNs were purified
by reversed-phase HPLC.
To evaluate the hybridization property of the Chol-dT fluorescence,
[
16]
melting experiments
were conducted using 8, 9, and their respective un-
ꢁ
ꢁ
modified ODNs 8 and 9 (Figure 2).
The melting temperature (T ) of the duplex 8/9, was higher than that
m
ꢁ
◦
ꢁ
◦
ꢁ
of the natural duplex 8 /9 (+2.4 C) and much higher than that of 8 /9 and
ꢁ
◦
8
/9 (+8.1 C and +8.8 C respectively) (Table 1).
FIGURE 2 Fluorescence melting curves at pH 7.0 (10 mM sodium phosphate buffer containing 200 mM
NaCl and 1 mM Na2EDTA). The concentration of each ODN was 0.5 µM.

7
88
A. Durand and T. Brown
TABLE 1 Fluorescence melting curves at pH 7.0 (10 mM sodium phosphate buffer
containing 200 mM NaCl and 1 mM Na2EDTA). Each Tm is an average of two
separate runs
buffer with NaCl 200 mM
ꢁ
ꢁ
ꢁ
ꢁ
ODN
Tm ( C)
8 /9
67.6
8 /9
8/9
61.2
8/9
70
◦
61.9
These results clearly illustrate a destabilization of the duplex with a
cholesterol moiety on one DNA strand, but stabilization when cholesterol
is present on both strands. We are planning further melting studies of the
duplex under various conditions.
REFERENCES
1
2
.
.
Seeman, N.C. At the crossroads of chemistry, biology, and materials: Structural DNA nanotechnol-
ogy. Chem. Biol. 2003, 10(12), 1151–1159.
3.
Seeman, N.C.; Wang, H.; Yang, X.P.; Liu, F.R.; Mao, C.D.; Sun, W.Q.; Wenzler, L.; Shen, Z.Y.; Sha,
R.J.; Yan, H.; Wong, M.H.; Sa-Ardyen, P.; Liu, B.; Qiu, H.X.; Li, X.J.; Qi, J.; Du, S.M.; Zhang, Y.W.;
Mueller, J.E.; Fu, T.J.; Wang, Y.L.; Chen, J.H. New motifs in DNA nanotechnology. Nanotechnol. 1998,
9(3), 257–273.
4
5
6
7
8
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Ding, B.Q.; Sha, R.J.; Seeman, N.C. Pseudohexagonal 2D DNA crystals from double crossover cohe-
sion. J. Am. Chem. Soc. 2004, 126(33), 10230–10231.
Chelyapov, N.; Brun, Y.; Gopalkrishnan, M.; Reishus, D.; Shaw, B.; Adleman, L. DNA triangles and
self-assembled hexagonal tilings. J. Am. Chem. Soc. 2004, 126(43), 13924–13925.
Liu, D.; Wang, M.S.; Deng, Z.X.; Walulu, R.; Mao, C.D. Tensegrity: Construction of rigid DNA trian-
gles with flexible four-arm DNA junctions. J. Am. Chem. Soc. 2004, 126(8), 2324–2325.
Yan, H.; Park, S.H.; Finkelstein, G.; Reif, J.H.; LaBean, T.H. DNA-templated self-assembly of protein
arrays and highly conductive nanowires. Science 2003, 301(5641), 1882–1884.
European Commission Leaflet. “EU nanotechnology research: Exploring the fundamentals,”
February 2005. Available at: http://cordis.europa.eu/search/index.cfm?fuseaction=proj.simple-
document&PJ RCN= 7522710&CFID= 9908140&CFTOKEN=76514940.
9.
Letsinger, R.L.; Chaturvedi, S.K.; Farooqui, F.; Salunkhe, M. Use of hydrophobic substituents in
controlling self-assembly of oligonucleotides. J. Am. Chem. Soc. 1993, 115(16), 7535–7536.
1
1
0. Vu, H.; Hill, T.S.; Jayaraman, K. Synthesis and properties of cholesteryl-modified triple-helix forming
oligonucleotides containing a triglycyl linker. Bioconjugate Chem. 1994, 5(6), 666–668.
1. Lehmann, T.J.; Serwe, M.; Caselmann, W.H.; Engels, J.W. Design and properties of hepatitis C virus
antisense oligonucleotides for liver specific drug targeting. Nucleosides Nucleotides & Nucleic Acids
2001, 20(4–7), 1343–1346.
12. Reed, M.W.; Lukhtanov, E.A.; Gorn, V.V.; Lucas, D.D.; Zhou, J.H.; Pai, S.B.; Cheng, Y.C.; Meyer, R.B.
Structure-activity-relationships of cytotoxic cholesterol-modified DNA duplexes. J. Medicinal Chem.
1995, 38(22), 4587–4596.
1
3. Bijsterbosch, M.K.; Manoharan, M.; Dorland, R.; Waarlo, I.H.E; Biessen, E.A.L.; van Berkel, T.J.C.
Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lac-
tosylated low-density lipoprotein. Biochem. Pharmacol. 2001, 62(5), 627–633.
1
1
1
4. Pfeiffer, I.; Hook, F. Bivalent cholesterol-based coupling of oligonucletides to lipid membrane as-
semblies. J. Am. Chem. Soc. 2004, 126(33), 10224–10225.
5. Benkoski, J.J.; Hook, F. Lateral mobility of tethered vesicle—DNA assemblies. J. Physical Chem. B
2005, 109(19), 9773–9779.
6. Darby, R.A.J.; Sollogoub, M.; McKeen, C.; Brown, L.; Risitano, A.; Brown, N.; Barton, C.; Brown, T.;
Fox, K.R. High throughput measurement of duplex, triplex and quadruplex melting curves using
molecular beacons and a LightCycler. Nucleic Acids Res. 2002, 30(9).

Oligonucleotides Containing Cholesterol Thymidine Monomer
SUPPLEMENTARY MATERIAL
789
1
H NMR spectra were measured at 400 MHz on a Bruker DPX400 spec-
13
trometer. C NMR spectra were measured at 100 MHz on a Bruker DPX400
spectrometer. Chemical shifts are given in ppm relative to tetramethylsilane,
and J values are given in Hz and are correct to within 0.5 Hz. Multiplicities of
13
C signals were determined using DEPT spectral editing technique and are
described as s (quaternary carbon), d (CH), t (CH ) and q (CH ). Assign-
2
3
1
1
31
ment was also aided by COSY ( H- H) and HMQC experiments. P NMR
spectra were recorded on a Bruker AC300 spectrometer at 121 MHz and
were externally referenced to 85 % phosphoric acid in D O. Low-resolution
2
mass spectra were recorded using electrospray technique on a Fisons VG
platform instrument in acetonitrile or a Waters ZMD quadrupole mass spec-
trometer in methanol. High-resolution mass spectra were recorded using
electrospray technique on a Bruker APEX III FT-ICR mass spectrometer in
acetonitrile or methanol. Infrared spectra were recorded on a Satellite FT-IR
using a Golden Gate adapter and WIN FIRST-lite software or using a Smart
Orbit adapter and OMNIC software. Absorptions are described as strong (s),
medium (m) and weak (w). Melting points were measured on a Gallenkamp
electrothermal melting point apparatus and are uncorrected. Elemental
analyses were performed by Medac Ltd using CE-440 and Carlo Erba ele-
mental analysers and the results were within ± 0.50 of the calculated values.
All reactions were carried out under inert atmosphere. Dichloromethane,
pyridine, DIPEA and triethylamine were dried over CaH , methanol was
2
dried over iodine and magnesium and THF over sodium and benzophe-
none.
12-(2,2,2-trifluoroacetamido)-N-(prop-2-ynyl)dodecanamide (1). 12-Amino-
dodecanoic acid (5.0 g, 23.22 mmol) was dissolved in MeOH (55 mL)
and triethylamine (10 mL) was added. The solution was stirred at rt and
ethyltrifluoroacetate (4.14 mL, 34.83 mmol) was added. The reaction was
complete after 22.5 hours. The solvents were removed in vacuo to give a
pale yellow solid which was then redissolved in distilled CH Cl (100 mL).
2
2
EDC (7.14 g, 37.26 mmol) and propargylamine (1.96 mL, 28.66 mmol)
were added to the solution which was stirred at rt for 24 hours. The
reaction was diluted with CH Cl , washed with saturated NaHCO solution,
2
2
3
then saturated KCl solution and dried (Na SO ), filtered and the solvents
2
4
removed in vacuo to give a pale yellow solid. The crude mixture was purified
by silica gel column chromatography (6:4 EtOAc:hexane) to give 1 as a
◦
−1
colourless solid (6.35 g, 79% overall): mp 91–92 C; ν
(neat)/cm 3315
max
(
m, NH), 3290 (m, NH), 2918 (m, CH ), 2851 (w, CH ), 1697 (s, CO),
2 2
1
644 (s, CO), 1570 (w), 1531 (m), 1470 (m, CH ), 1448 (w, CH ), 1416
2 2
1
(w), 1373 (w), 1207 (s, CF ), 1179 (s, CF ), 719 (s); H NMR (400 MHz,
3 3
CDCl ): δ 6.43 (s, 1H), 5.65 (s, 1H), 4.04 (dd, J = 5.0, 2.5 Hz, 2H), 3.35
3
H
(
q, J = 6.9 Hz, 2H), 2.22 (t, J = 2.5 Hz, 1H), 2.18 (t, J = 7.5 Hz, 2H), 1.59

7
90
A. Durand and T. Brown
1
3
(
7
m, 4H), 1.28 (m, 14H); C NMR (100 MHz, CDCl ): δ 172.8, 157.3, 79.8,
3
C
+
1.6, 40.1, 36.6, 29.4, 29.4, 29.3, 29.3, 29.1 29.0, 26.7, 25.6; LRMS [ES ]:
+
+
+
m/z 349 (M+H , 80%), 371 (M+Na , 100%); HRMS [ES ]: m/z found
+
3
5
49.2093 (M+H) C H F N O requires 349.2098; Anal calculated: C,
17
28
3
2
2
8.61; H, 7.81; N, 8.04. Found: C, 58.78; H, 7.84; N, 7.95.
ꢁ
ꢁ
ꢁ
ꢁ
3
,5 -Di-O-acetyl-5-iodo-2 -deoxyuridine (3). 5-Iodo-2 -deoxyuridine (500
mg, 1.41 mmol) was dissolved in pyridine (3 mL) and acetic anhydride (40
µL, 4.24 mmol) was added. The reaction was stirred under argon for 16.5
hours at rt, then the pyridine was removed and co-evaporated with toluene
in vacuo. The crude mixture was purified by column chromatography (6:4
◦
EtOAc:hexane) to give 3 as a colourless solid (610 mg, 99%): mp 156–157 C;
−
1
ν
(neat)/cm 3236 (m, NH), 1736 (s, CO), 1673 (s, CO), 1362 (s, CH),
max
1
1
235 (s, C-O), 1194 (s, C-O), 1106 (s, C-O); H NMR (400 MHz, CDCl ):
3
δ 9.20 (s, 1H), 7.96 (s, 1H), 6.28 (dd, J = 8.3, 5.6 Hz, 1H), 5.23 (dt, J =
H
6
.0, 2.0 Hz, 1H), 4.40 (dd,J = 12.6, 3.0 Hz, 1H), 4.33 (dd,J = 12.6, 3.0
Hz, 1H), 4.29 (dd, J = 6.0, 3.0 Hz, 1H), 2.54 (ddd, J = 14.1, 5.5, 2.0, 1H),
2
1
.19 (m, 1H), 2.20 (s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, CDCl ): δ
3 C
70.7, 170.5, 160.0, 150.2, 144.1, 85.8, 83.0, 74.4, 69.3, 64.2, 38.6, 21.4, 21.2;
+
+
+
LRMS [ES ]: m/z461 (M+Na , 20%); HRMS [ES ]: m/z found 460.9823
+
(
3
M+Na) C H IN O Na requires 460.9816; Anal calculated: C, 35.63; H,
13
15
2
7
.45; N, 6.39. Found: C, 35.60; H, 3.38; N, 6.31.
ꢁ
ꢁ
ꢁ
5
-(12-amino-N-(prop-2-ynyl)dodecanamido)-3 ,5 -di-O-acetyl-2 -deoxyuridine (4).
1.0 g, 2.87 mmol) was diluted in ethanolic methylamine 33% (25 mL,
01 mmol) and the reaction mixture was stirred at rt for 24 hours. The
(
2
solvent was removed in vacuo and dried overnight under high vacuum
to give a white solid (929 mg), which was used without further purifica-
tion. Compound 3 (193 mg, 0.44 mmol) was dissolved in DMF (2 mL) to
which the deprotected amine (244 mg, 0.97 mmol), triethylamine (2.46 mL,
17.62 mmol) and CuI (34 mg, 0.18 mmol) were added. The solution
was stirred in the dark, at rt, under argon for 10 min before adding
tetrakis(triphenylphosphine)palladium (102 mg, 0.88 mmol). The reaction
had gone to completion after 30 hours. The solvents were removed in
vacuo, the residue dissolved in a mixture of CH Cl and MeOH, filtered on
2
2
celite and the solvents were removed in vacuo. A methanolic solution of the
crude material was pre-adsorbed onto silica and purified by column chro-
matography (8:2 EtOAc:hexane) to give 4 as brown syrup (238 mg, 96%):
−
1
ν
max(neat)/cm 3254 (w, NH), 2925 (m, CH ), 2844 (w),1710 (s, CO),
2
1
9
651 (s, CO), 1456 (m), 1362 (m, CH), 1228 (s, C–O), 1101 (m), 1023 (m),
1
77 (C–O), 776 (m), 603 (m), 564 (m); H NMR (400 MHz, CDCl ): δ 7.93
3
H
(
s, 1H), 6.18 (dd, J = 7.7, 6.2 Hz, 1H), 5.24 (dt, J = 6.6, 2.6 Hz, 1H), 4.26–
4
1
4
.37 (m, 3H), 4.10 (s, 2H), 2.89 (m, 2H), 2.47 (ddd, J = 14.6, 6.2, 2.7 Hz,
H), 2.15–2.23 (m, 1H), 2.17 (m, 2H), 2.14 (s, 3H), 2.09 (s, 3H), 1.62 (m,
13
H), 1.31 (m, 14H); C NMR (100 MHz, CDCl ): δ 177.0, 173.2, 145.6,
3
C

Oligonucleotides Containing Cholesterol Thymidine Monomer
791
1
2
01.6, 88.4, 85.2, 76.7, 66.1, 42.0, 39.6, 38.0, 31.6, 31.5, 31.5, 31.3, 31.3, 29.7,
+
+
+
8.5, 27.9, 22.1, 21.9; LRMS [ES ]: m/z 563 (M+H , 30%); HRMS [ES ]:
+
m/z found 563.3066 (M+H) C H N O requires 563.3075.
28
43
4
8
ꢁ
ꢁ
5
-(12-Cholesteryloxycarbonylamino-N-(prop-2-ynyl)dodecanamido)-3 ,5 -di-O-
ꢁ
acetyl-2 -deoxyuridine (5). A solution of cholesteryl chloroformate (133 mg,
0
.30 mmol) in distilled CH Cl (0.6 mL) was added to a stirred solution of
2 2
4
(139 mg, 0.25 mmol) in DMF (1 mL) with DIPEA (50 µL, 0.30 mmol).
◦
The solution was stirred at 0 C, under an argon atmosphere for 2 hours and
then at rt for a further 2 hours. The solvents were removed in vacuo and the
residue was dissolved in CH Cl , washed with citric acid 10%, saturated KCl
2
2
solution, dried (Na SO ), filtered and the solvents removed in vacuo. The
2
4
crude mixture was purified by column chromatography (EtOAc:hexane 3:2
◦
−1
to 4:1) to give 5 as a yellow solid (77 mg, 33%): mp 132 C; νmax(neat)/cm
273 (w, NH), 2924 (m, CH ), 2845 (m, CH ), 2358 (w, C-C alkyne), 1695
3
2
3
(
1
s, CO), 1628 (s), 1542 (m), 1459 (m, CH ), 1361 (m, CH ), 1224 (s, C-O),
2 3
1
039 (m), 953 (m, C-O), 776 w), 604 (w); H NMR (400 MHz, CDCl ):
3
δ 9.06 (s, 1H), 7.80 (s, 1H), 6.26 (dd, J = 7.5, 6.0 Hz, 1H), 6.06 (br s, 1H),
H
5
4
.36 (m, 1H), 5.23 (dt, J = 6.5, 3.0 Hz, 1H), 4.64 (br s, 1H), 4.48 (br s, 1H),
.36 (m, 2H), 4.28 (dd, J = 6.5, 3.0 Hz, 1H), 4.23 (d, J = 5.02 Hz, 2H), 3.13
(
3
m, 2H), 2.54 (ddd, J = 14.1, 6.0, 2.5 Hz, 1H), 2.20–2.25 (m, 3H), 2.16 (s,
13
H), 2.11 (s, 3H), 1.47 (m, 4H), 1.25 (m, 14H), 1.07–2.56 (br m, 43H); C
NMR (100MHz, CDCl ): δ 173.0, 170.4, 149.1, 142.3, 140.0, 122.6, 100.1,
3
C
8
3
2
5.8, 82.8, 74.1, 63.8, 56.8, 56.3, 50.2, 42.5, 39.9, 39.7, 38.7, 38.4, 37.1, 36.7,
6.6, 36.3, 35.09, 32.0, 32.0, 30.1, 30.0, 29.8, 29.6, 29.5, 29.4, 29.4, 29.4,
8.3, 28.1, 26.9, 25.6, 24.4, 24.0, 22.9, 22.7, 21.2, 21.0, 20.1, 19.5, 18.9, 12.0;
+
+
+
LRMS [ES ]: m/z 997 (M+Na , 100%); HRMS [ES ]: m/z found 997.6244
+
(
8
M+Na) C H N O Na requires 997.6236; Anal calculated: C, 68.96; H,
56
86
4
10
.89; N, 5.74. Found: C, 69.29; H, 9.01; N, 5.51.
ꢁ
ꢁ
5
-(12-Cholesteryloxycarbonylamino-N-(prop-2-ynyl)dodecanamido)-5 -O-(4,4 -
ꢁ
dimethoxytrityl)-2 -deoxyuridine (6). Compound 5 (800 mg, 0.82 mmol) was
diluted in potassium carbonate solution at 0.4 mol.L in MeOH:H O 4:1
−
1
2
(13 mL, 5.20 mmol). The reaction mixture was stirred at rt for 1.75 hours
then diluted in CH Cl . DOWEX 50WX8-400 (pyridinium form) was
2
2
added and the solution was stirred until pH = 7, filtered and the solvents
were evaporated and co-evaporated with pyridine in vacuo to give a solid
(
(
891 mg) which was used without further purification. This compound
653 mg, 0.73 mmol) was dissolved in CH Cl (5 mL) to which 3 mL of
2
2
ꢁ
pyridine was added followed by 4,4 -dimethoxytrityl chloride (298 mg, 0.88
mmol) in small portions. The reaction was stirred for 5 hours and 5 mL
of MeOH was added to the solution. The solvents were evaporated and
co-evaporated with toluene in vacuo and the crude mixture was purified
by column chromatography (EtOAc:hexane 4:1, 1% pyridine) to give 6
◦
−1
as a yellow solid (358 mg, 45%): mp 104 C; νmax(neat)/cm 3346 (w,

7
92
A. Durand and T. Brown
NH), 3056 (w, CH Ar), 2925 (m, CH ), 2844 (m, CH ), 2357 (w, C–C
2
3
alkyne), 1687 (s, CO), 1601 (m), 1503 (s, C=C Ar), 1446 (s, CH ), 1283
2
(
s), 1242 (s, C–O), 1172 (s), 1172 (s), 1086 (m), 1025 (s, C–O), 825 (m),
1
7
8
1
4
23 (m), 694 (m), 576 (s); H NMR (400 MHz, CDCl ): δ 9.40 (s, 1H),
3 H
.19 (s, 1H), 6.83–7.44 (m, 13H), 6.32 (br t, J = 6.5 Hz, 1H), 6.18 (br s,
H), 5.53 (br s, 1H), 5.37 (br s, 1H), 4.83 (br s, 1H), 4.69 ( br s, 1H),
.11 (s, 1H), 3.87 (d, J = 5.5 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 3H), 3.43
(
d, J = 9 Hz, 1H), 3.33 (d, J = 8 Hz, 1H), 3.13 (br s, 2H), 2.51 (m, 1H),
2
.22–2.36 (m, 3H), 1.52 (m, 4H), 1.26 (m, 14H), 0.97–6.16 (br m, 50H);
13
C NMR (100 MHz, CDCl ): δ 207.0, 172.7, 158.8 , 147.5, 144. 7, 143.3,
3
C
1
1
5
3
2
40.0, 135.7, 135.6, 130.2, 130.2, 130.1, 129.3, 129.2 128.3, 128.2, 128.1,
27.9, 127.2, 122.6, 113.3, 86.3, 85.9, 81.6, 56.8, 56.3, 56.3, 55.4, 55.4, 55.3,
4.0, 50.2, 42.5, 41.7, 41.1, 41.0, 40.9, 39.9, 39.7, 38.7, 38.2, 37.1, 36.7, 36.4,
6.4, 36.3, 36.3, 35.9, 32.0, 31.0, 30.1, 30.0, 29.6, 29.6, 29.5, 29.4, 28.4, 28.3,
8.1, 26.9, 26.9, 25.7, 25.6, 24.4, 24.0, 24.0, 22.9, 22.7, 21.2, 19.5, 18.9, 12.0;
+
+
LRMS [ES ]: m/z 1216 (M+Na , 40%); Anal calculated: C, 73.46; H, 8.44;
N, 4.69. Found: C, 73.16; H, 8.46; N, 4.59.
ꢁ
ꢁ
5
-(12-Cholesteryloxycarbonylamino-N-(prop-2-ynyl)dodecanamido)-5 -O-(4,4 -
ꢁ
ꢁ
dimethoxytrityl)-2 -deoxyuridine-3 -O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite
7). 2-Cyanoethyl-N ,N -diisopropylchlorophosphine (61 µL, 0.28 mmol)
(
was added dropwise to a degassed solution of 6 (298 mg, 0.25 mmol)
in THF (1.5 mL) with DIPEA (109 µL, 0.62 mmol). After stirring at rt
for 0.5 hour, the solution was transferred via cannula under argon to a
separating funnel containing degassed ethyl acetate. The mixture was
washed with saturated KCl solution, dried (Na SO ) and the solvent was
2
4
removed in vacuo. Purification by column chromatography under argon
pressure (EtOAc: Hexane 4:1, 0.5% pyridine) gave 7 as a colourless air
1
sensitive foam (284 mg, 82%): H NMR (400 MHz, d -DMSO): δ 11.61 (br
6
H
s, 1H), 7.92 (br s, 1H), 6.86–7.40 (m, 14H), 6.96 (br s, 1H), 6.88 (dd, J =
1
3
3
3.6, 7.0 Hz, 1H), 5.31(br s, 1H), 4.29 (br t, J = 11 Hz, 1H), 4.02 (s, 1H),
.93 (d, J = 5.0 Hz, 1H), 3.90 (d, J = 5.0 Hz, 1H),3.74 (s, 3H), 3.73 (s,
H), 3.62 (m, 2H), 3.52 (m, 2H), 3.19 (d, J = 7.5 Hz, 2H), 3.14 (d, J = 7.5
Hz, 2H), 2.92 (q, J = 6.5 Hz, 2H), 2.75 and 2.64 (t, J = 5.8 Hz, 2H), 2.40
(m, 1H), 2.23 (m, 1H), 1.30–1.51 (m, 4H), 1.21 (m, 14H), 0.64–2.59 (br
31
+
m, 59H); P NMR (162 MHz, d -DMSO)δ 148.5, 148.9; LRMS [ES ]: m/z
6
p
+
1216 (M+Na , 40%).
Oligonucleotide Synthesis
All oligonucleotides were synthesised on an Applied Biosystems 394
automated DNA/RNA synthesiser using a standard 0.2 µmole phospho-
ramidite cycle of acid-catalysed detritylation, coupling, capping and iodine

Oligonucleotides Containing Cholesterol Thymidine Monomer
793
oxidation. Stepwise coupling efficiencies and overall yields were determined
by the automated trityl cation conductivity monitoring facility and were
>
98.0%. Chol-T phosphoramidite monomer was dissolved in anhydrous
−
1
CH Cl (100 mg.mL ) coupled for 360 seconds rather than the standard
2
2
25 seconds. Standard A, G, C and T DNA phosphoramidites, solid supports
and additional reagents were purchased from Link Technologies or Applied
Biosystems Ltd. Cleavage of the oligonucleotides from the solid support was
achieved by exposure to concentrated ammonia solution (30 min at rt) fol-
◦
lowed by heating in conc. aqueous ammonia, 55 C for 5 hours in a sealed
tube.
Purification of oligonucleotides was carried out by reversed phase HPLC
on a Gilson system using an ABI Aquapore column (C8), 8 mm × 250 mm,
˚
pore size 300 A. The system was controlled by Gilson 7.12 software and the
following protocol was used: Run time 30 mins, flow rate 4 mL per min,
binary system, gradient: Time in mins (% buffer B);0 (0); 3(0); 5(20); 21
(100); 25(100); 27 (0); 30(0). Buffers for unmodified oligonucleotides: Elu-
tion buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M ammonium
acetate with 25% acetonitrile pH 7.0. Buffers for modified oligonucleotides:
Elution buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M
ammonium acetate with 60% acetonitrile pH 7.0. Elution of oligonu-
cleotides was monitored by ultraviolet absorption at 295 nm. After HPLC
purification oligonucleotides were desalted using disposable NAP 10
Sephadex columns (Pharmacia), aliquoted into eppendorf tubes and stored
◦
at –20 C. Purified oligonucleotides containing the synthesised monomer
were then analysed by MALDI-TOF MS using a ThermoBioAnalysis Dy-
namo MALDI-TOF mass spectrometer in positive ion mode using internal
[1]
T standards.
n
ODN
Sequence
d(5 -3 )
Molecular ion
expected(M+H ) found(M+H )
Molecular ion
ꢁ
ꢁ
+
+
Code
8
9
ACCAAGGTGCTATCGTCG
CGACGATAGCACCTTGGT
6149.6
6149.6
6149.6
6150.7
T = Chol dT.
Fluorescence melting experiments
A 5 µM stock solution of each oligonucleotide was prepared in distilled
water and the experiments were performed using a Roche LightCycler in
a volume of 20 µL containing 0.5 µM of each strand, and the appropriate

7
94
A. Durand and T. Brown
concentration of NaCl (200 mM, 400 mM or 600 mM NaCl) with 10 mM
sodium phosphate, 1 mM Na EDTA at pH = 7.0 and 2 µL of SyberGreen
2
◦
(
2
1:1000 v:v). The complexes were denaturated by heating to 95 C at a rate of
0 C/sec and were maintained at this temperature for 5 mins before cool-
ing to 30 C at 0.1 C/sec. Samples were then held at 30 C for 5 mins before
melting again by heating to 95 C at 0.1 C/sec. Data were collecting dur-
ing annealing and melting steps and compared. The lightcycler has one
excitation source (488 nm) and the changes in fluorescence emission were
measured at 520 nm. Each experiment was performed in duplicate.
◦
◦
◦
◦
◦
◦
REFERENCE
1.
Langley, G.J.; Herniman, J.M.; Davies, N.L.; Brown, T. Simplified sample preparation for the analysis
of oligonucleotides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Rapid Commun. Mass Spectrom. 1999, 13(17), 1717–1723.