Diamondoid-modified DNA

Article abstract of DOI:10.1039/c1ob05929g

We prepared novel C5-modified triphosphates and phosphoramidites with a diamondoid functionally linked to the nucleobase. Using primer extension experiments with different length templates we investigated whether the modified triphosphates were enzymatica

Full text of DOI:10.1039/c1ob05929g

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PAPER
Diamondoid-modified DNA†
Yan Wang,a Boryslav A. Tkachenko,b Peter R. Schreiner*b and Andreas Marx*a
Received 9th June 2011, Accepted 2nd August 2011
DOI: 10.1039/c1ob05929g
We prepared novel C5-modified triphosphates and phosphoramidites with a diamondoid functionally
linked to the nucleobase. Using primer extension experiments with different length templates we
investigated whether the modified triphosphates were enzymatically incorporated into DNA and
whether they were further extended. We found that all three modified nucleotides can be incorporated
into DNA using a single-nucleotide incorporation experiment, but only partially using two templates
that demand for multiple incorporation of the modified nucleotides. The modified phosphoramidites
were introduced into oligonucleotides utilizing DNA synthesizer technology. The occurring
oligonucleotide structures were examined by circular dichroism (CD) and melting temperature (Tm)
measurements and were found to adapt similar helix conformations as their unmodified counterparts.
Introduction
Deoxyribonucleic acid (DNA) represents the complete genetic
database of nature and thus contains all information necessary
for synthesis of ribonucleic acid (RNA) and proteins. These are
required for the development of a biological organism and the
Fig. 1 Structures of the selected diamondoids and their nomenclature: a)
metabolism in the cells. Since the discovery of DNA it has been
in constant focus of molecular biology and biochemical research.
There is a growing interest in the synthesis of functionalized DNA,
and an important aspect is that by modifying the structure of
the double stranded DNA it must not significantly compromise
duplex formation. Most appropriate for functionalization is the
part of the nucleobase that is not involved in hydrogen bonds of
Watson–Crick base pairing.1–3 In addition, when utilizing sterically
demanding moieties a sufficiently long linker should be used to
reduce the interactions between DNA and the added structural
moiety. Particularly attractive, sterically highly demanding build-
ing blocks are the so-called diamondoids (nanodiamonds), which
are a member of the nanoscale carbon materials family, consisting
of face-fused carbon cage (adamantane) repeating units that are
superimposable on the diamond lattice. Their terminal bonds are
saturated by hydrogen, leading to a unique molecular hierarchy of
the form C4n+6H4n+12, where n = 1, 2, 3, . . . specifies the polymantane
order (Fig. 1).4–6 Diamondoids display a variety of geometries,
offering a large tool kit of atomically-perfect building blocks of
different shapes and sizes. Following isolation from petroleum
deposits,7 the diamondoids can be selectively derivatized by
adamantane, b) diamantane, and c) triamantane.
substituting various functional groups for surface hydrogens,8
and their derivatives have found widespread applications from
materials science9 to medical applications.10
Here we show, for the first time, that diamondoids (Fig. 1)
can be introduced as a lipophilic and bulky functional group at
the C5-position of the pyrimidine moiety and then incorporated
into DNA by chemical and enzymatic means. It has been shown
that modifications at this position do not significantly interfere
with Watson–Crick base pairing.11–20 Here we report the synthesis
of diamondoid-modified triphosphates and phosphoramidites
(Fig. 2). We found that these nucleotides can be incorporated into
aDepartment of Chemistry, University of Konstanz, Universita¨tsstrasse
10, 78457, Konstanz, (Germany). E-mail: andreas.marx@uni-konstanz.de;
Fax: (+49) 7531-88-5140; Tel: (+49) 7531-88-5139
bInstitute of Organic Chemistry, Justus-Liebig University, Heinrich-
Buff-Ring 58, 35392, Gießen, (Germany). E-mail: prs@org.chemie.uni-
giessen.de; Fax: (+49) 641-9934–309; Tel: (+49) 641-9934-300
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/c1ob05929g
Fig. 2 Diamondoid modified triphosphates and phosphoramidites.
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DNA by DNA polymerases and also by automated solid phase
DNA synthesis. The conformation of the resulting diamondoid-
modified DNA was investigated by thermal denaturation
studies (Tm measurements) as well as circular dichroism (CD)
spectroscopy.
Results and discussion
Synthesis of modified triphosphates
Scheme 2 a) CuI, [Pd(PPh3)4], Et3N, 1-O-diamondoidyl-4-O-propargyl
triethylene glycol 4, DMF, in microwave, 50 ◦C, 20 min, 8a: 66%, 8b: 70%,
8c: 63%; b) N-ethyldiisopropylamine, 2-cyanoethyl-N,N-diisopro-
pylchloro-phosphoramidite, CH2Cl2, 0 ◦C 30 min, then rt, 3 h, 2a: 70%,
2b: 75%, 2c: 80%.
In order to place the bulky diamondoid modification away from
the nucleobase we choose a triethylene glycol linkage (Fig. 2)
that has been shown to be accepted by DNA polymerases.16
First we planned the synthesis of 1-O-diamantoidyl-4-O-propargyl
triethylene glycol 4. For this purpose, the bromides 3a,b,c21–23 were
prepared and heated in the presence of triethylamine with 1-O-
propargyl triethylene glycol24,25 for 5 h at 180 ◦C and converted into
1-O-diamondoidyl-4-O-propargyl triethylene glycol 4a,b,c. The
compounds 4a,b,c were then coupled to 5-iodo-2¢-deoxyuridine
5 by using a standard protocol for the Sonogashira reaction16,26–28
to obtain nucleosides 6a,b,c. These nucleosides were then con-
verted into the corresponding triphosphates 1a,b,c by using an
appropriate method for phosphorylation29–31 (Scheme 1).
triphosphates 1 into a growing DNA strand by substitution of the
natural counterpart. We carried out primer extension experiments
using a 5¢-32P-labeled 23-nucleotide primer and several templates.
The reactions were analysed by denaturing polyacrylamide gel
(PAGE). Visualisation was performed by phosphorimaging.
First we investigated a 35-nucleotide template bearing a single A
residue at position 27 calling for incorporation of a thymidine ana-
logue. We used KlenTaq DNA-polymerase, N-terminally short-
ened form of Taq DNA polymerase.37 Lacking dTTP KlenTaq
DNA polymerase wild-typeincorporated a mismatched nucleotide
opposite position 27 and then paused.
Reactions including all four natural dNTPs gave rise to full-
length products. Full-length product was also observed when natu-
ral dTTP was replaced by one of the modified triphosphates 1 (Fig.
3a). In the following experiments we used two different templates
(69 nucleotides) to code for a modified dTTP every fourth and
second position, respectively. For template calling for a modified
nucleotide in every fourth position we found that the DNA
polymerase was able to form full-length products in the presence
of three natural dNTPs and each of the three modified dTTPs 1
(Fig. 3b). Upon substitution of natural TTP by 1a,b,c the
full-length DNA products migrated more slowly in the gel in
Scheme 1 a) 1-O-propargyl triethylene glycol, Et3N, 180 ◦C, 5 h, 4a:
83%, 4b: 76%, 4c: 79%; b) CuI, [Pd(PPh3)4], Et3N, 1-O-diamondoidyl-
4-O-propargyl triethylene glycol 4, DMF, rt, 24 h, 6a: 75%, 6b: 75%,
6c: 79%; c) proton sponge (1,8-bis(dimethylamino)naphthalene), POCl3,
PO(OMe)3, 0 ◦C, then (Bu3NH)2H2P2O7, nBu3N, then triethylammonium
bicarbonate (TEAB) buffer, 1a: 6%, 1b: 5%, 1c: 5%.
Synthesis of modified phosphoramidites
The procedure for the synthesis of C5-modified 2¢-deoxyuridine-
3¢-O-phosphoramidites 2 was similar to that for the triphos-
phates. 5-Iodo-5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxy-uridine 7 was
prepared according to literature protocols32,33 and was coupled to
4a,b,c by using the Sonogashira reaction26–28 to create nucleosides
8a,b,c. The reactions were conducted in a microwave leading to
higher yields compared to reactions performed without microwave
assistance. The compounds 8a,b,c were then converted into the cor-
responding phosphoramidites 2a,b,c using a standard method34–36
(Scheme 2). The phosphoramidites 2 were purified rapidly after
reaction and stored under argon atmosphere at -20 ◦C.
Fig. 3 The primer extension experiments by KlenTaq DNA polymerase.
For a) and b) Lanes 1: 5¢-32P-labeled 23-nucleotide primer strand; lanes 2:
primer template complex including dATP, dCTP, dGTP; lanes 3: same as
lanes 1 including TTP; lanes 4: same as lanes 1 including 1a; lanes 5: same
as lanes 1 including 1b; lanes 6: same as lanes 1 including 1c.
Incorporation of diamondoid-functionalised nucleotides using
primer extension
The primer extension reactions should provide information
whether DNA polymerases are able to incorporate the modified
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Table 1 Overview of the synthesized oligonucleotidesa
Molar masses
Name
Sequence
Calculated molar mass/(g mol-1)
Found molar mass/(g mol-1)
ON_Ref
5¢-GGT CTT AGC TAT-3¢
5¢-ATA GCT AAG ACC-3¢
5¢-GGT CTT* AGC TAT-3¢
5¢-GGT CTT* AGC TAT-3¢
5¢-GGT CTT* AGC TAT-3¢
3751.5
3638.5
3957.9
4010.0
4062.1
3750.6
3637.4
3957.2
4008.1
4060.3
ON_Com
ON_Adam
ON_Diam
ON_Triam
a The oligonucleotides were prepared on a DNA synthesizer. The modifications of the oligonucleotides were inserted using 2a, 2b and 2c. The single
strands ON1_Adam, ON1_Diam and ON1_Triam contain the modification of adamantane, diamantane and triamantane.
comparison with the unmodified full-length product (Fig. 3a, b).
We suppose that this property is based on the increased steric
demand and higher molecular weight of the modified entities;
similar effects have been reported before.13 For the template calling
for a modified nucleotide in every second position we found no full-
length products and only fragments (data not shown) indicating
that the modifications prevent further modification density.
Oligonucleotide synthesis
In order to investigate the duplex properties of diamondoid-
modified DNA, the phosphoramidites 2a,b,c were used to synthe-
size modified oligonucleotides using a DNA synthesizer.38,39 The
Fig. 4 CD-spectra of unmodified DNA (—) and adamantane modified
unmodified oligonucleotide40 (ON_Ref) was used as reference and
the oligonucleotide40 (ON_Com) as the complementary strand.
The oligonucleotides ON_Adam, ON_Diam and ON_Triam con-
tained an adamantane-, diamantane-, and triamantane-modified
nucleotide. The oligonucleotides were prepared using standard
methods.41–47 An overview of the synthesized oligomers is shown
in Table 1.
DNA (
), diadamantane modified DNA (
) and triadamantane
modified DNA (
). The CD spectra show the characteristic profile of
a B-DNA with a positive maximum at about 275 nm and a minimum at
about 250 nm. Compared to the unmodified oligonucleotides the curve of
the modified ones do not show significant differences.
Circular dichroism (CD) and melting temperature (Tm)
measurements
The synthesized oligonucleotides were purified by preparative
PAGE and further characterized by thermal denaturation studies
(melting temperature (Tm) measurements) as well as by CD
spectroscopy. To determine the influence of the modifications on
the duplex DNA conformation, CD spectra were measured of
the respective modified strand hybridized to the complementary
standard ON_Com.
The recorded CD spectra (Fig. 4) showed the characteristic
curve of a B-DNA with a positive maximum at about 275 nm
and a minimum at about 245 nm for all duplexes. In comparison
with the unmodified oligonucleotides the curves of the modified
are very much alike. This indicates that the modifications of
the investigated diamandoid-modified thymidines are on one
hand well accommodated in the major groove, and on the other
hand flexible enough without interfering with the overall B-DNA
conformation.
The Tm measurements (Fig. 5) indicate duplex stability of
the modified oligonucleotides. Tm values of the modified double
strands are lower than for the corresponding unmodified DNA
strand (54.7 ◦C). The measured Tm values of adamantane,
diamantane, and triamantane modified duplexes are 4.4, 4.9, and
5.2 ◦C lower, respectively. The decrease of the Tm values indicate
a minor destabilization of these modified double strands.
Fig. 5 Melting temperature curves of unmodified DNA (—), adamantane
modified DNA (
), diamantane modified DNA (
) and triaman-
tane modified DNA (
). The observed Tm for the unmodified 12 nt
DNA was 54.7 ◦C. The three modified DNA adamantane, diamantane and
triamantane show Tm values of 50.3 ◦C, 49.8 ◦C and 49.5 ◦C, respectively.
Conclusions
A straightforward synthesis of diamondoid-modified thymidines
was developed. The corresponding triphosphates 1a,b,c are rec-
ognized as substrates for incorporation into the growing DNA
strand. Enzymatic synthesis of DNA strands that contain mod-
ifications at every fourth position was feasible as well. The cor-
responding phosphoramidites 2a,b,c were assembled into oligo-
nucleotides using a DNA synthesizer. CD spectroscopy and
melting temperature (Tm) measurements indicate that the
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B-conformation of DNA double helix is tolerated by the
diamondoid-modified nucleotides, and the decrease of the Tm
values show only slight destabilization of the duplex by these
modifications. Hence, diamondoid-modified DNA can be utilized
to exploit some of the attractive features of these diamond-like
molecules by their targeted assembly. Such features include inter
alia pronounced electron emission.9
1-O-(1-adamantyl)-4-O-propargyl triethylene glycol (4a)
The reaction was carried out with 1-bromo-adamantane 3a
(430 mg, 2.0 mmol), 1-O-propargyl triethylene glycol (3.8 g,
20 mmol) and triethylamine (0.80 mL, 6.0 mmol). Purification:
silica gel (n-hexane/EtOAc 2 : 1). Rf: 0.43 (n-hexane/EtOAc
2 : 1). Yield: 535 mg (83%). 1H-NMR (400 MHz, CDCl3): d
4.15 (2H, d, 4J = 2.4 Hz, OCH2CCH), 3.67–3.58 (8H, m,
OCH2CH2OCH2CH2O), 3.56–3.50 (4H, m, adam-OCH2CH2),
4
2.40 (1H, t, J = 2.4 Hz, CCH), 2.09 (3H, m, adam-H-3,5,7),
Experimental section
1.69 (6H, m, adam-H-2,8,9), 1.50–1.52 (6H, m, adam-H-4,6,10).
13C-NMR (101 MHz, CDCl3): d 79.7, 74.5, 72.2, 71.3, 70.6, 70.4,
General
+
69.1, 59.3, 58.4, 41.5, 36.5, 30.5. HRMS: m/z: calcd for C19H31O4
([M+H]+): 323.2217, found: 323.2208.
All reagents are commercially available and were used without
further purification. Anhydrous solvents were obtained from
Sigma–Aldrich, stored over molecular sieves and used without
further purification. All synthetic reactions were performed
under inert atmosphere. 5-Iodo-2¢-deoxyuridine was purchased
from Carbosynth. NMR spectra were recorded using Bruker
Avance 400 (1H: 400, 13C: 101, 32P: 162 MHz) and DRX 600
(1H: 600, 13C: 151, 32P: 243 MHz) spectrometers. Chemical shifts
are given in parts per million, tetramethylsilane was used as
internal standard. Electrospray ionization ion trap (ESI-IT) mass
spectra were recorded on a Bruker Daltonics Esquire 3000+
in positive or negative mode with a flow rate of 5 mL/min.
Flash chromatography was done using Merck silica gel G60
1-O-(4-diamantyl)-4-O-propargyl triethylene glycol (4b)
The reaction was carried out with 4-bromo-diamantane 3b
(219 mg, 0.82 mmol), 1-O-propargyl triethylene glycol (1.54 g,
8.2 mmol) and triethylamine (0.35 mL, 2.46 mmol). Purification:
silica gel (n-hexane/EtOAc 2 : 1). Rf: 0.49 (n-hexane/EtOAc
2 : 1). Yield: 234 mg (76%). 1H-NMR (400 MHz, CDCl3): d
4.19 (2H, d, 4J = 2.4 Hz, OCH2CCH), 3.69–3.64 (8H, m,
OCH2CH2OCH2CH2O), 3.59–3.57 (4H, m, diam-OCH2CH2),
4
2.42 (1H, t, J = 2.4 Hz, CCH), 1.92 (m, 3H, diam-H-2,6,12),
1.78–1.76 (1H, m, diam-H-9), 1.72–1.69 (15H, m, diam-H-
1,3,5,7,8,10,11,13,14). 13C-NMR (101 MHz, CDCl3): d 79.8, 74.6,
71.7, 71.4, 70.7, 70.5, 69.3, 59.7, 58.5, 41.9, 39.7, 37.3, 36.8, 25.7.
(40–63 mm) and Merck pre-coated plates (silica gel 60 F254
)
were used for TLC. For medium pressure liquid chromatography
(MPLC), a Bu¨chi unit with a Bu¨chi controller C-620, two
pumps C-605, a UV monitor C-630 (l = 254 nm) and fraction
collector C-660 was used. For reverse phase chromatography of
nucleosides, a D40-RP-18 ready-to-use column (Go¨tec, 25–40 mm)
was used. For reverse phase chromatography of nucleotides, a
HRMS: m/z: calcd for C23H35O4 ([M+H]+): 375.2530, found:
+
375.2536.
1-O-(9-triamantyl)-4-O-propargyl triethylene glycol (4c)
R
The reaction was carried out with 9-bromo-triamantane 3c
(255 mg, 0.80 mmol), 1-O-propargyl triethylene glycol (1.5 g,
8.0 mmol) and triethylamine (0.30 mL, 2.4 mmol). Purification:
silica gel (n-hexane/EtOAc 2 : 1). Rf: 0.54 (n-hexane/EtOAc
2 : 1). Yield: 270 mg (79%). 1H-NMR (400 MHz, CDCl3): d
4.19 (2H, d, 4J = 2.4 Hz, OCH2CCH), 3.69–3.65 (8H, m,
OCH2CH2OCH2CH2O), 3.58–3.56 (4H, m, triam-OCH2CH2),
2.42 (1H, t, 4J = 2.4 Hz, CCH), 1.86–1.82 (3H, m, triam-H-
7,11,15), 1.70–1.58 (14H, m, triam-H-2,3,4,5,6,12,13,14,16,18),
1.37–1.30 (6H, m, triam-H-8,10,17). 13C-NMR (101 MHz,
CDCl3): d 79.8, 74.6, 72.4, 71.4, 70.7, 70.5, 69.3, 59.7, 58.5, 48.8,
46.1, 45.3, 41.8, 40.3, 38.1, 37.6, 37.5, 35.7, 35.0, 34.5, 27.2.
310-25 LiChroprepꢀ RP-18 ready-to-use column (Merck, 40–
63 mm) was used. Purification of triphosphates was performed on
a BioLogic DuoFlow System (Bio-Rad Laboratories) with DEAE
SephadexTM A-25 (GE Healthcare Bio-Sciences AB) column using
a linear gradient of TEAB-buffer (0.1–1.0 M, pH = 7.5). Desoxynu-
cleotides were purchased from Fermentas, oligonucleotides from
Metabion (purified twice by HPLC) and T4 polynucleotide kinase
from New England Biolabs. CD spectra was measured on a Jasco
J-715 instrument in phosphate-buffer (20 mM K2HPO4/KH2PO4,
pH = 7.0, 1.0 M NaCl) at 25 ◦C. Melting curves were recorded on
a Varian Cary 100 bio UV/Vis Spectrophotometer. The sample
(same as for CD spectra) contained 1 ¥ phosphate-buffer (20 mM
K2HPO4/KH2PO4, pH = 7.0, 1.0 M NaCl).
HRMS: m/z: calcd for C27H39O4 ([M+H]+): 427.2843, found:
+
427.2839.
General procedure for 1-O-diamondoidyl-4-O-propargyl
General procedure for nucleosides (6a–c)
triethylene glycol (4a–c)
5-Iodo-2¢-deoxyuridine
5 (1.0 equiv) and copper(I) iodide
1-O-diamondoidyl-4-O-propargyl triethylene glycol 4 was synthe-
sized by the reaction of bromodiamondoid 3 (1.0 equiv) and
1-O-propargyl triethylene glycol (10 equiv) at 180 ◦C in the
presence of triethylamine (3.0 equiv) for 5 h. After cooling to
room temperature ethyl acetate (40 mL) was added. The solution
was washed with 2 M hydrochloric acid (2 ¥ 30 mL) and brine
(2 ¥ 30 mL). The organic layer was dried over magnesium sulfate
and the solvent evaporated under reduced pressure. The residue
was purified by flash chromatography.
(0.2 equiv) were dissolved in anhydrous DMF. With stir-
ring the corresponding 1-O-diamondoidyl-4-O-propargyl triethy-
lene glycol 4 (3.0 equiv), tetrakis(triphenylphosphine)palladium
(0.1 equiv) and triethylamine (3.0 equiv) were added. The mixture
was protected from light and stirred at rt for 24 h. The mixture
was combined with sat. sodium bicarbonate solution (10 mL) and
extracted with dichloromethane (4 ¥ 10 mL). The organic layers
were washed with 2 M hydrochloric acid (2 ¥ 10 mL) and with
brine (2 ¥ 10 mL), this was dried over magnesium sulfate and the
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solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography and RP-MPLC.
(5–100% acetonitrile). The product eluted at 64% acetonitrile to
1
yield 51.6 mg (79%). Rf: 0.55 (DCM/MeOH 10 : 1). H-NMR
3
(400 MHz, CDCl3): d 8.42 (1H, s, H-6), 6.21 (1H, t, J = 6.0
Hz, H-1¢), 4.58–4.54 (1H, m, H-3¢), 4.41–4.31 (2H, m, CCCH2O),
3.99–3.98 (1H, m, H-4¢), 3.95–3.84 (2H, m, H-5¢), 3.79–3.66
(8H, m, CH2OCH2CH2OCH2), 3.62–3.56 (4H, m, triam-OCH2,
CH2OCH2CC), 2.46–2.39 (1H, m, H-2¢), 2.33–2.27 (1H, m, H-2¢),
1.87–1.82 (3H, m, triam-H-7,11,15), 1.71–1.58 (14H, m, triam-H-
2,3,4,5,6,12,13,14,16,18), 1.37–1.28 (6H, m, triam-H-8,10,17). 13C-
NMR (101 MHz, CDCl3): d 162.0, 149.6, 144.8, 99.1, 89.2, 87.5,
86.2, 78.2, 73.0, 71.3, 70.6, 70.4, 70.1, 69.2, 61.3, 59.5, 59.3, 48.7,
46.0, 45.3, 41.7, 41.2, 40.2, 38.0, 37.6, 37.4, 35.7, 35.0, 34.4, 27.2.
5-[12-(Adamantyl-1-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine (6a)
The reaction was carried out with 5-iodo-2¢-deoxyuridine 5
(229 mg, 0.65 mmol), copper(I) iodide (24.8 mg, 0.13 mmol),
1-O-(1-adamantyl)-4-O-propargyl triethylene glycol 4a (420 mg,
1.30 mmol), tetrakis(triphenylphosphine)palladium (75.1 mg,
65 mmol) and triethylamine (0.27 mL, 1.95 mmol) in 5 mL
anhydrous DMF. Purification: silica gel (DCM/MeOH 20 : 1)
and RP-MPLC (RP-18, 25–40 mm) using a linear gradient of
water/acetonitrile (5–100% acetonitrile). The product eluted at
55% acetonitrile to yield 267 mg (75%). Rf: 0.43 (DCM/MeOH
HRMS: m/z: calcd for C37H49N2O11 ([M+HCO2]-): 697.3342,
-
found: 697.3342.
1
10 : 1). H-NMR (400 MHz, CDCl3): d 8.29 (1H, s, H-6), 6.18
General procedure for 5¢-O-(4,4¢-dimethoxytrityl)nucleosides
(8a–c)
(1H, t, 3J = 6.0 Hz, H-1¢), 4.52–4.51 (1H, m, H-3¢), 4.38–4.30 (2H,
m, CCCH2O), 4.00–3.99 (1H, m, H-4¢), 3.90–3.80 (2H, m, H-
5¢), 3.72–3.64 (8H, m, CH2OCH2CH2OCH2), 3.60–3.55 (4H, m,
adam-OCH2, CH2OCH2CC), 2.44–2.40 (1H, m, H-2¢), 2.31–2.25
(1H, m, H-2¢), 2.12 (3H, m, Adam-H-3,5,7), 1.72 (6H, m, adam-H-
2,8,9), 1.63–1.54 (6H, m, adam-H-4,6,10). 13C-NMR (101 MHz,
CDCl3): d 162.3, 149.7, 144.7, 99.1, 89.3, 87.6, 86.2, 78.0, 72.8,
71.2, 70.5, 70.4, 69.2, 61.6, 59.2, 41.5, 41.1, 36.5, 30.6. HRMS: m/z:
calcd for C29H41N2O11- ([M+HCO2]-): 593.2716, found: 593.2724.
5-Iodo-5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxyuridine 7 (1.0 equiv)
and copper(I) iodide (0.2 equiv) were dissolved in anhy-
drous DMF. With stirring the corresponding 1-O-diamondoidyl-
4-O-propargyl triethylene glycol
4 (3.0 equiv), tetrakis-
(triphenylphosphine)palladium (0.1 equiv) and triethylamine (3.0
equiv) were added. The mixture was stirred in synthesis microwave
(InitiatorTM, Biotage) at 50 ◦C for 20 min. The mixture was
combined with sat. sodium bicarbonate solution (10 mL) and
extracted with dichloromethane (4 ¥ 10 mL). The organic layers
were washed with 2 M hydrochloric acid (2 ¥ 10 mL) and with
brine (2 ¥ 10 mL), this was dried over magnesium sulfate and the
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography and RP-MPLC.
5-[12-(Diamantyl-4-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine (6b)
The reaction was carried out with 5-iodo-2¢-deoxyuridine 5
(35.3 mg, 0.10 mmol), copper(I) iodide (3.8 mg, 20 mmol), 1-
O-(4-diamantyl)-4-O-propargyl triethylene glycol 4b (74.9 mg,
0.20 mmol), tetrakis(triphenylphosphine)palladium (11.6 mg,
10 mmol) and triethylamine (42 mL, 0.30 mmol) in 1 mL anhydrous
DMF. Purification: silica gel (DCM/MeOH 20 : 1) and RP-MPLC
(RP-18, 25–40 mm) using a linear gradient of water/acetonitrile
(5–100% acetonitrile). The product eluted at 60% acetonitrile
to yield 45.1 mg (75%). Rf: 0.51 (DCM/MeOH 10 : 1). 1H-
NMR (400 MHz, CDCl3): d 8.38 (1H, s, H-6), 6.20 (1H, t,
3J = 5.2 Hz, H-1¢), 4.55–4.54 (1H, m, H-3¢), 4.40–4.31 (2H, m,
CCCH2O), 3.99 (1H, m, H-4¢), 3.93–3.84 (2H, m, H-5¢), 3.69–
3.64 (8H, m, CH2OCH2CH2OCH2), 3.61–3.57 (4H, m, diam-
OCH2, CH2OCH2CC), 2.44–2.40 (1H, m, H-2¢), 2.32–2.28 (1H,
m, H-2¢), 1.92 (3H, m, diam-H-2,6,12), 1.78–1.77 (1H, m, diam-H-
9), 1.71–1.68 (15H, m, diam-H-1,3,5,7,8,10,11,13,14). 13C-NMR
(101 MHz, CDCl3): d 162.0, 149.6, 144.8, 99.1, 89.3, 87.6, 86.2,
78.1, 72.2, 71.3, 70.6, 70.4, 70.3, 69.2, 61.4, 59.6, 59.3, 41.8,
5-[12-(Adamantyl-1-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine (8a)
The reaction was carried out with 5-iodo-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine
7 (197 mg, 0.30 mmol),
copper(I) iodide (11.4 mg, 60 mmol), 1-O-(1-adamantyl)-
4-O-propargyl triethylene glycol 4a (145 mg, 0.50 mmol),
tetrakis-(triphenylphosphine)palladium (34.7 mg, 30 mmol) and
triethylamine (130 mL 1.5 mmol) in 2 mL anhydrous DMF.
Purification: silica gel (EtOAc/n-hexane, 10 : 1 + 1% Et3N)
and RP-MPLC (RP-18, 25–40 mm) using a linear gradient of
acetonitrile/water (5–100% acetonitrile). The product eluted at
40% acetonitrile to yield 168.2 mg (66%). Rf: 0.32 (EtOAc/n-
hexane, 5 : 1). 1H-NMR (400 MHz, Acetone-d6): d 8.08 (1H,
s, H-6), 7.53–7.51 (2H, m, Ar–H), 7.41–7.39 (4H, m, Ar–H),
7.36–7.32 (2H, m, Ar–H), 7.26–7.23 (1H, m, Ar–H), 6.92–6.89
(4H, m, Ar–H), 6.28 (1H, t, 3J = 6.8 Hz, H-1¢), 4.61–4.58
(1H, dd, 3J = 4.4 Hz, 3J = 7.6 Hz, H-3¢), 4.11–4.09 (3H, m,
H-4¢, CCCH2O), 3.80 (6H, s, OCH3), 3.57–3.47 (12H, m,
OCH2CH2OCH2CH2OCH2CH2O), 3.42–3.30 (2H, m, H-5¢),
-
41.1, 39.7, 37.3, 36.8, 25.7. HRMS: m/z: calcd for C33H45N2O11
([M+HCO2]-): 645.3029, found: 645.3027.
5-[12-(Triamantyl-9-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine (6c)
3
2
The reaction was carried out with 5-iodo-2¢-deoxyuridine 5
(35.3 mg, 0.10 mmol), copper(I) iodide (3.8 mg, 20 mmol), 1-
O-(9-triamantyl)-4-O-propargyl triethylene glycol 4c (85.3 mg,
0.20 mmol), tetrakis(triphenylphosphine)palladium (11.6 mg,
10 mmol) and triethylamine (42 mL, 0.30 mmol) in 1 mL anhydrous
DMF. Purification: silica gel (DCM/MeOH 20 : 1) and RP-MPLC
(RP-18, 25–40 mm) using a linear gradient of water/acetonitrile
2.42–2.39 (2H, dd, J = 4.4 Hz, J = 6.4 Hz, H-2¢), 2.09 (3H,
m, adam-H-3,5,7), 1.72 (6H, m, adam-H-2,8,9), 1.66–1.58 (6H,
m, adam-H-4,6,10). 13C-NMR (101 MHz, Acetone-d6): d 161.9,
159.6, 150.3, 146.0, 143.9, 136.9, 136.6, 131.0, 128.9 128.8, 127.7,
114.1, 99.8, 89.9, 87.6, 86.4, 78.6, 72.3, 72.2, 72.0, 71.3, 71.0, 69.8,
64.7, 60.1, 59.3, 55.6, 42.3, 41.8, 37.2, 31.4. HRMS: m/z: calcd
for C49H57N2O11-([M - H]-): 849.3968, found: 849.3963.
7486 | Org. Biomol. Chem., 2011, 9, 7482–7490
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5-[12-(Diamantyl-4-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine (8b)
in vacuum, dissolved in trimethyl phosphate, and cooled to
0 ◦C. Freshly distilled phosphoryl chloride (20 equiv) was added
dropwise with stirring. The mixture was stirred at 0 ◦C for 6 h.
A 0.5 M solution of bis(tri-n-butylammonium)pyrophosphate in
anhydrous DMF (20 equiv) and tri-n-butylamine (40 equiv) were
added simultaneously to the mixture. After 5 min, 1 M aqueous
triethylammonium bicarbonate (TEAB buffer, pH 7.5) was added
and the aqueous layer was washed with EtOAc (3 ¥ 2 mL). The
aqueous layer was concentrated in vacuo. and the resulting residue
purified by ion-exchange chromatography [DEAE SephadexTM A-
25, linear gradient of TEAB buffer (0.1 M to 1 M, 1000 mL),
flow 2 mL min-1] and further purified by RP-MPLC (RP-18, 40–
63 mm) using a gradient of 5% (200 mL), 20% (200 mL) and 40%
(200 mL) acetonitrile in 50 mM aq. triethylammonium acetate
(TEAA buffer, pH 7.0).
The reaction was carried out with 5-iodo-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine 7 (175 mg, 0.30 mmol), copper(I)
iodide (10.0 mg, 54 mmol), 1-O-(4-diamantyl)-4-O-propargyl
triethylene glycol 4b (150 mg, 0.40 mmol), tetrakis(triphenyl-
phosphine)palladium (31 mg, 27 mmol) and triethylamine
(0.1 mL, 0.80 mmol) in 2 mL anhydrous DMF. Purification:
silica gel (EtOAc/n-hexane, 10 : 1 + 1% Et3N) and RP-MPLC
(RP-18, 25–40 mm) using a linear gradient of acetonitrile/water
(5–100% acetonitrile). The product eluted at 45% acetonitrile to
yield 169.1 mg (70%). Rf: 0.36 (EtOAc/n-hexane, 5 : 1). 1H-NMR
(400 MHz, Acetone-d6): d 8.08 (1H, s, H-6), 7.53–7.51 (2H, m,
Ar–H), 7.41–7.39 (4H, m, Ar–H), 7.36–7.32 (2H, m, Ar–H), 7.26–
3
7.23 (1H, m, Ar–H), 6.92–6.89 (4H, m, Ar–H), 6.28 (1H, t, J =
6.8 Hz, H-1¢), 4.61–4.58 (1H, dd, 3J = 4.4 Hz, 3J = 7.6 Hz, H-3¢),
4.12–4.09 (3H, m, H-4¢; CCCH2O), 3.80 (6H, s, OCH3), 3.57–3.47
(12H, m, OCH2CH2OCH2CH2OCH2CH2O), 3.42–3.31 (2H, m,
H-5¢), 2.42–2.39 (2H, m, H-2¢), 1.92 (3H, m, diam-H-2,6,12),
1.74–1.69 (16H, m, diam-H-1,3,5,7,8,9,10,11,13,14). 13C-NMR
(101 MHz, Acetone-d6): d 161.9, 159.7, 150.3, 146.0, 143.9, 136.9,
136.6, 131.0, 128.9 128.8, 127.6, 114.1, 99.8, 89.9, 87.6, 86.4, 78.6,
72.4, 72.0, 71.5, 71.3, 71.0, 69.9, 64.7, 60.5, 59.3, 55.6, 42.7, 41.8,
5-[12-(Adamantyl-1-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine-5¢-triphosphate (1a)
The reaction was carried out with 5-[12-(Adamantyl-1-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-2¢-deoxyuridine 6a (45 mg, 82 mmol),
1,8-bis(dimethylamino)naphthalene (proton sponge, 43 mg,
0.20 mmol), phosphoryl chloride (200 mL), solution of bis(tri-
n-butylammonium)pyrophosphate (3.5 mL, 1.7 mmol) and tri-
n-butylamin (0.8 mL, 3.3 mmol) in 2.0 mL trimethylphosphate.
Yield: 6.0 mg of triethylammonium salt (6% estimated by UV
absorption). 1H-NMR (600 MHz, D2O): d 8.19 (1H, s, H-6),
-
40.5, 38.0, 37.7, 26.6. HRMS: m/z: calcd for C53H61N2O11 ([M -
H]-): 901.4281, found: 901.4282.
5-[12-(Triamantyl-9-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine (8c)
3
6.27 (1H, t, J = 6.4 Hz, H-1¢), 4.64–4.61 (1H, m, H-3¢), 4.48
(2H, m, CCCH2O), 4.25–4.15 (3H, m, H-4¢, H-5¢), 3.82–3.67
(12H, m, OCH2CH2OCH2CH2OCH2CH2O), 3.21–3.18 (24H, m,
(HN(CH2CH3)3)4 ), 2.42–2.39 (2H, m, H-2¢), 2.11 (m, 3H, adam-
The reaction was carried out with 5-iodo-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine
7 (185 mg, 0.28 mmol),
+
copper(I) iodide (11.0 mg, 56 mmol), 1-O-(9-triamantyl)-
4-O-propargyl triethylene glycol 4c (180 mg, 0.42 mmol),
tetrakis-(triphenylphosphine)palladium (33 mg, 28 mmol) and
triethylamine (0.12 mL, 0.84 mmol) in 2 mL anhydrous DMF.
Purification: silica gel (EtOAc/n-hexane, 10 : 1 + 1% Et3N)
and RP-MPLC (RP-18, 25–40 mm) using a linear gradient of
acetonitrile/water (5–100% acetonitrile). The product eluted at
50% acetonitrile to yield 170.1 mg (63%). Rf: 0.40 (EtOAc/n-
H-3,5,7), 1.75 (6H, m, adam-H-2,8,9), 1.69–1.60 (6H, m, adam-H-
4,6,10), 1.30 (36H, t, 3J = 6.8 Hz, (HN(CH2CH3)3)4 ). 31P-NMR
+
(162 MHz, D2O): d -10.6 (1P, m, Pg ), -11.7 (1P, m, Pa), -22.7
-
(1P, m, Pb). HRMS: m/z: calcd for C28H42N2O18P3 ([M - H]-):
787.1651, found: 787.1651.
1
hexane, 5 : 1). H-NMR (400 MHz, Acetone-d6): d 8.07 (1H, s,
5-[12-(Diamantyl-4-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine-5¢-triphosphate (1b)
H-6), 7.53–7.51 (2H, m, Ar–H), 7.42–7.39 (4H, m, Ar–H), 7.36–
7.32 (2H, m, Ar–H), 7.26–7.23 (1H, m, Ar–H), 6.92–6.90 (4H, m,
The reaction was carried out with 5-[12-(Diamantyl-4-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-2¢-deoxyuridine 6b (33 mg, 55 mmol),
1,8-bis(dimethylamino)naphthalene (proton sponge, 21.5 mg,
0.10 mmol), phosphoryl chloride (100 mL), solution of bis(tri-
n-butylammonium)pyrophosphate (2.2 mL, 1.1 mmol) and tri-
n-butylamin (0.5 mL, 2.2 mmol) in 2.0 mL trimethylphosphate.
Yield: 3.5 mg of triethylammonium salt (5% estimated by UV
3
Ar–H), 6.28 (1H, t, J = 6.8 Hz, H-1¢), 4.59–4.58 (1H, m, H-3¢),
4.12–4.09 (3H, m, H-4¢; CCCH2O), 3.80 (6H, s, OCH3), 3.56–3.47
(12H, m, OCH2CH2OCH2CH2OCH2CH2O), 3.42–3.31 (2H,
m, H-5¢), 2.42–2.39 (2H, m, H-2¢), 1.86–1.81 (3H, m, triam-H-
7,11,15), 1.72–1.60 (14H, m, triam-H-2,3,4,5,6,12,13,14,16,18),
1.39–1.29 (6H, m, triam-H-8,10,17). 13C-NMR (101 MHz,
Acetone-d6): d 161.9, 159.7, 150.3, 146.0, 143.9, 136.9, 136.6,
131.0, 130.0 128.8, 127.7, 114.1, 99.8, 89.9, 87.6, 86.4, 78.6,
72.4, 72.0, 71.4, 71.3, 71.0, 69.9, 64.7, 60.5, 59.3, 55.6, 49.7,
46.9, 46.0, 42.5, 41.8, 41.2, 38.7, 38.3, 36.3, 35.9, 35.3, 28.1.
HRMS: m/z: calcd for C57H65N2O11- ([M - H]-): 953.4594, found:
953.4596.
1
absorption). H-NMR (600 MHz, D2O): d = 8.19 (1H, s, H-6),
3
6,28 (1H, t, J = 6.4 Hz, H-1¢), 4.65–4.63 (1H, m, H-3¢), 4.47
(2H, m, CCCH2O), 4.22–4.09 (3H, m, H-4¢, H-5¢), 3.82–3.65
(12H, m, OCH2CH2OCH2CH2OCH2CH2O), 3.23–3.19 (24H, m,
+
(HN(CH2CH3)3)4 ), 2.42–2.39 (2H, m, H-2¢), 1.93 (3H, m, diam-
H-2,6,12), 1.72–1.67 (m, 16H, diam-H-1,3,5,7,8,9,10,11,13,14),
+
1.28 (36H, t, 3J = 6.8 Hz, (HN(CH2CH3)3)4 ). 31P-NMR
General procedure for nucleotides (1a–c)
(162 MHz, D2O): d -10.8 (1P, m, Pg ), -11.5 (1P, m, Pa), -23.1
-
The nucleosides 6 (1.0 equiv) and 1,8-bis(dimethylamino)-
(1P, m, Pb). HRMS: m/z: calcd for C32H46N2O18P3 ([M - H]-):
naphthalene (proton sponge, 1.5 equiv) were dried overnight
839.1964, found: 839.1963.
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Org. Biomol. Chem., 2011, 9, 7482–7490 | 7487
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5-[12-(Triamantyl-9-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-2¢-
deoxyuridine-5¢-triphosphate (1c)
31P-NMR (162 MHz, Acetone-d6): d 148.4, 148.2. ESI-MS: m/z:
1086.4 [M + Cl]-.
The reaction was carried out with 5-[12-(Triamantyl-9-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-2¢-deoxyuridine 6c (34 mg, 52 mmol),
1,8-bis(dimethylamino)naphthalene (proton sponge, 21.5 mg,
0.10 mmol), phosphoryl chloride (95 mL), solution of bis(tri-n-
butylammonium)pyrophosphate (2.0 mL, 1.0 mmol) and tri-n-
butylamin (0.5 mL, 2.1 mmol) in 2.0 mL trimethylphosphate.
Yield: 3.2 mg of triethylammonium salt (5% estimated by UV
absorption). 1H-NMR (600 MHz, D2O): d 8.20 (1H, s, H-
6), 6.26 (1H, t, 3J = 6,4 Hz, H-1¢), 4.65–4.62 (1H, m, H-
3¢), 4.44 (2H, m, CCCH2O), 4.23–4.11 (3H, m, H-4¢, H-5¢),
3.79–3.61 (12H, m, OCH2CH2OCH2CH2OCH2CH2O), 3.26–
5-[12-(Diamantyl-4-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine-3¢-(cyanoethyl-N,N¢-
diisopropyl)phosphoramidite (2b)
The reaction was carried out with 5-[12-(Diamantyl-4-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-dimethoxytrityl)-2¢-deo-
xyuridine 8b (90.3 mg, 0.10 mmol), N-ethyldiisopropylamine
(51 mL, 0.30 mmol) and 2-cyanoethyl-N,N¢-diisopropyl-
chlorophosphor-amidite (45 mL, 0.20 mmol) in 5 mL anhydrous
dichloromethane. Purification: silica gel (EtOAc/n-hexane, 7 : 1
+ 1% Et3N). Yield: 83.0 mg (75%). Rf: 0.62 (EtOAc/n-hexane,
5 : 1). 1H-NMR (400 MHz, Acetone-d6): d 8.10 (0.5H, s, H-6), 8.08
(0.5H, s, H-6), 7.54–7.51 (2H, m, Ar–H), 7.43–7.39 (4H, m, Ar–
H), 7.37–7.32 (2H, m, Ar–H), 7.27–7.24 (1H, m, Ar–H), 6.93–6.90
(4H, m, Ar–H), 6.31–6.26 (1H, m, H-1¢), 4.77–4.71 (1H, m, H-3¢),
4.26–4.20 (1H, m, H-4¢) 4.15–4.07 (2H, CCCH2O), 3.80 (6H, s,
OCH3), 3.57–3.45 (12H, m, OCH2CH2OCH2CH2OCH2CH2O),
3.44–3.36 (2H, m, H-5¢), 2.80–2.63 (4H, m, OCH2CH2CN),
2.61–2.52 (2H, m, H-2¢), 1.92 (3H, m, diam-H-2,6,12), 1.75–
1.69 (16H, m, diam-H-1,3,5,7,8,9,10,11,13,14), 1.28–1.10 (14H,
m, N(CH(CH3)2)2). 13C-NMR (101 MHz, Acetone-d6): d 161.8,
159.7, 150.2, 145.9, 143.9, 136.7, 136.5, 131.0, 128.9, 128.8, 127.7,
114.1, 99.9, 90.0, 87.6, 86.3, 78.5, 72.0, 71.5, 71.3, 71.2, 71.0, 69.8,
64.3, 60.5, 59.7, 59.5, 59.2, 55.6, 42.6, 40.7, 40.5, 38.0, 37.6, 26.6,
24.9, 24.8, 20.8, 20.7. 31P-NMR (162 MHz, Acetone-d6): d 148.4,
148.2. ESI-MS: m/z: 1138.8 [M + Cl]-.
+
3.22 (24H, m, (HN(CH2CH3)3)4 ), 2.43–2.39 (2H, m, H-2¢),
1.87–1.85 (3H, m, triam-H-7,11,15), 1.72–1.60 (20H, m, triam-
3
H-2,3,4,5,6,8, 10,12,13,14,16,17,18), 1.30 (36H, t, J = 6.8 Hz,
+
(HN(CH2CH3)3)4 ). 31P-NMR (162 MHz, D2O): d -10.6 (1P, m,
Pg ), -11.6 (1P, m, Pa), -22.9 (1P, m, Pb). HRMS: m/z: calcd for
C36H50N2O18P3 ([M - H]-): 891.2277, found: 891.2277.
-
General procedure for Phosphoramidite (2a–c)
A solution of 5-[12-(diamondoidyloxyl)-(4,7,10-trioxadodec-1-
ynyl)]-5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxyuridine 8 (1.0 equiv)
in anhydrous dichloromethane was stirred at
0
◦C. N-
ethyldiisopropylamine (3.0 equiv) and 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (2.0 equiv) were added drop-
wise with stirring. The mixture was stirred at 0 ◦C for 15 min.
The reaction was allowed to stir at room temperature for 3 h
and quenched with 5 mL anhydrous methanol. The residue was
washed with ice-cold water (2 ¥ 20 mL) and once with ice-cold
brine. The organic layer was dried over magnesium sulfate and the
solvent was evaporated under reduced pressure. The residue was
then purified by flash chromatography.
5-[12-(Triamantyl-9-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine-3¢-(cyanoethyl-N,N¢-
diisopropyl)phosphoramidite (2c)
5-[12-(Adamantyl-1-oxyl)-(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-
dimethoxytrityl)-2¢-deoxyuridine-3¢-(cyanoethyl-N,N¢-
diisopropyl)phosphoramidite (2a)
The reaction was carried out with 5-[12-(Triamantyl-9-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-dimethoxytrityl)-2¢deo-
xyuridine 8c (95.5 mg, 0.10 mmol), N-ethyldiisopropylamine
(51 mL, 0.30 mmol) and 2-cyanoethyl-N,N¢-diisopropyl-
chlorophosphor-amidite (45 mL, 0.20 mmol) in 5 mL anhydrous
dichloromethane. Purification: silica gel (EtOAc/n-hexane, 5 : 1
+ 1% Et3N). Yield: 92.0 mg (80%). Rf: 0.65 (EtOAc/n-hexane,
The reaction was carried out with 5-[12-(Adamantyl-1-oxyl)-
(4,7,10-trioxadodec-1-ynyl)]-5¢-O-(4,4¢-dimethoxytrityl)-2¢-de-
oxyuridine 8a (85.1 mg, 0.10 mmol), N-ethyldiisopropylamine
(51 mL, 0.30 mmol) and 2-cyanoethyl-N,N¢-diisopropyl-
chlorophosphor-amidite (45 mL, 0.20 mmol) in 5 mL anhydrous
dichloromethane. Purification: silica gel (EtOAc/n-hexane, 10 : 1
+ 1% Et3N). Yield: 74.0 mg (70%). Rf: 0.59 (EtOAc/n-hexane,
5 : 1). 1H-NMR (400 MHz, Acetone-d6): d 8.07 (0.9H, s, H-6), 8.06
(0.1H, s, H-6), 7.53–7.51 (2H, m, Ar–H), 7.42–7.39 (4H, m, Ar–
H), 7.36–7.32 (2H, m, Ar–H), 7.27–7.23 (1H, m, Ar–H), 6.92–6.90
(4H, m, Ar–H), 6.30–6.27 (1H, m, H-1¢), 4.75–4.70 (1H, m, H-3¢),
4.21–4.19 (1H, m, H-4¢) 4.12–4.11 (2H, CCCH2O), 3.80 (6H, s,
OCH3), 3.57–3.48 (12H, m, OCH2CH2OCH2CH2OCH2CH2O),
3.42–3.36 (2H, m, H-5¢), 2.80–2.75 (4H, m, OCH2CH2CN), 2.63–
2.50 (2H, m, H-2¢), 2.09 (3H, m, adam-H-3,5,7), 1.72 (6H, m,
adam-H-2,8,9), 1.66–1.58 (6H, m, adam-H-4,6,10), 1.26–1.10 (m,
14H, N(CH(CH3)2)2). 13C-NMR (101 MHz, Acetone-d6): d 161.8,
159.7, 150.2, 145.9, 143.9, 136.7, 136.5, 131.0, 128.9, 128.8, 127.7,
114.1, 99.9, 90.0, 87.6, 86.3, 78.6, 72.2, 72.0, 71.3, 71.2, 71.0, 69.8,
64.3, 60.1, 59.2, 55.6, 42.3, 40.8, 37.2, 31.4, 24.9, 24.8, 20.8, 20.7.
1
5 : 1). H-NMR (400 MHz, Acetone-d6): d 8.10 (0.2H, s, H-6),
8.07 (0.8H, s, H-6), 7.53–7.51 (2H, m, Ar–H), 7.43–7.39 (4H, m,
Ar–H), 7.36–7.32 (2H, m, Ar–H), 7.27–7.23 (1H, m, Ar–H), 6.93–
6.90 (4H, m, Ar–H), 6.31–6,27 (1H, m, H-1¢), 4.77–4.69 (1H, m,
H-3¢), 4.27–4.19 (1H, m, H-4¢) 4.17–4.07 (CCCH2O), 3.80 (6H, s,
OCH3), 3.57–3.46 (12H, m, OCH2CH2OCH2CH2OCH2CH2O),
3.44–3.36 (2H, m, H-5¢), 2.80–2.75 (4H, m, OCH2CH2CN), 2.60–
2.52 (2H, m, H-2¢), 1.86–1.81 (3H, m, triam-H-7,11,15), 1.75–
1.59 (14H, m, triam-H-2,3,4,5,6,12,13,14,16,18), 1.39–1.29 (6H,
m, triam-H-8,10,17), 1.21–1.10 (14H, m, N(CH(CH3)2)2). 13C-
NMR (101 MHz, Acetone-d6): d 161.8, 159.7, 150.2, 145.9, 143.9,
136.7, 136.5, 131.0, 128.9, 128.8, 127.7, 114.1, 99.9, 90.0, 87.6, 86.3,
78.5, 72.3, 72.0, 71.3, 71.2, 71.0, 69.8, 64.3, 60.4, 59.7, 59.5, 59.3,
55.6, 49.7, 46.8, 45.9, 42.5, 41.1, 40.8, 38.7, 38.3, 36.2, 35.9, 35.3,
28.1, 24.9, 24.8, 20.8, 20.7. 31P-NMR (162 MHz, Acetone-d6): d
148.4, 148.2. ESI-MS: m/z: 1191.0 [M + Cl]-.
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Primer extension
contained 1 ¥ phosphate-buffer (20 mM K2HPO4/KH2PO4, pH =
7.0, 1.0 M NaCl). The duplex DNA samples were heated to
A primer extension reaction (20 mL) contained: 1 ¥ KlenTaq
reaction buffer (50 mM Tris · HCl pH 9.2, 16 mM (NH4)2SO4,
2.5 mM mgCl2, 0.1% Tween 20), 32P-labeled primer (150 nM),
template (200 nM), dTDTP (100 mM each), dATP, dCTP, dGTP
(100 mM each), DNA-polymerase (KlenTaq wild-type, 50 nM
each). First primer and template were annealed in 1 ¥ reaction
buffer by heating the probes to 95 ◦C for 2 min and stepwise cooling
to 0 ◦C. Afterwards the primer template complex, nucleotides
and DNA polymerase were incubated at 60 ◦C for 30 min. The
reactions were quenched by the addition of PAGE gel-loading
buffer (45 mL, 80% formamide, 20 mm EDTA, 0.1% bromophenol
blue, 0.1% xylene cyanole FF) and the product mixture was
analysed by 12% denaturing polyacrylamide gel. Visualization was
performed by phosphorimaging.
◦
◦
80 C for 2 min and cooled with a gradient of 1.0 C s-1 to the
final temperature of 25 ◦C prior to the measurements. Data were
taken from three individual cooling/heating cycles. The melting
temperatures (Tm values in ◦C) were calculated using the first
negative derivative of intensity over temperature.
Acknowledgements
We thank A. Baccaro for assistance and Dr K.-H. Jung for his
comments during manuscript preparation and Jeremy E. P. Dahl
for supplying triamantane.
Notes and references
Primer: BRAF-23C (23mer)
5¢-GAC CCA CTC CAT CGA GAT TTC TC-3¢
Template: BRAF-35C (35mer)
3¢-CTG GGT GAG GTA GCT CTA AAG AGG GCA CGG
TCG GC-5¢
Template: SO-69-NNNA (69mer)
3¢-CTG GGT GAG GTA GCT CTA AAG AGA CTT ACG
GAC TGA CGC ATT TAC TTA GCC ATT CAT TTA TCT
AGG GAT-5¢
1 Review: S. Weisbrod and A. Marx, Chem. Commun., 2008, 5675–5685.
2 G. Giller, T. Tasara, B. Angerer, K. Mu¨hlegger, M. Amacker and H.
Winter, Nucleic Acids Res., 2003, 31, 2630.
3 (a) O. Thum, S. Ja¨ger and M. Famulok, Angew. Chem., Int. Ed., 2001,
40, 3990; (b) S. Obeid, A. Baccaro, W. Welte, K. Diederichs and A.
Marx, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 21327.
4 Reviews: H. Schwertfeger, A. A. Fokin and P. R. Schreiner, Angew.
Chem., 2008, 120, 1038; H. Schwertfeger, A. A. Fokin and P. R.
Schreiner, Angew. Chem., Int. Ed., 2008, 47, 1022.
5 A. A. Fokin, B. A. Tkachenko, P. A. Gunchenko, D. V. Gusev and P.
R. Schreiner, Chem.–Eur. J., 2005, 11, 7091.
6 (a) P. R. Schreiner, N. A. Fokina, B. A. Tkachenko, H. Hausmann,
M. Serafin, J. E. P. Dahl, R. M. K. Carlson, S. G. Liu and A. A.
Fokin, J. Org. Chem., 2006, 71, 6709; (b) P. R. Schreiner, A. A. Fokin,
B. A. Tkachenko, E. Vass, M. Olmstead, D. Bla¨ser, R. Boese, H. P.
Reisenauer, J. E. P. Dahl and R. M. K. Carlson, J. Am. Chem. Soc.,
2009, 131, 11292.
Template: SO-69-NA (69mer)
3¢-CTG GGT GAG GTA GCT CTA AAG AGA CAT ACA
GAC AGA CAT ACA GAT ACA GAT ACA GAT ACA GAT
ACA GAT-5¢
7 J. E. Dahl, S. G. Liu and R. M. K. Carlson, Science, 2002, 299, 96
Admantane, diamantane, and triamantane are also available through
chemical synthesis, see strategies and references in ref. 4.
8 (a) B. A. Tkachenko, N. A. Fokina, L. V. Chernish, J. E. P. Dahl, S. G.
Liu, R. M. K. Carlson, A. A. Fokin and P. R. Schreiner, Org. Lett.,
2006, 8, 1767; (b) A. A. Fokin, T. E. Shubina, P. A. Gunchenko, S.
D. Isaev, A. G. Yurchenko and P. R. Schreiner, J. Am. Chem. Soc.,
2002, 124, 10718; (c) P. R. Schreiner, A. A. Fokin, O. Lauenstein, Y.
Okamoto, T. Wakita, C. Rinderspacher, G. H. Robinson, J. K. Vohs
and C. F. Campana, J. Am. Chem. Soc., 2002, 124, 13348.
Oligonucleotide synthesis
The oligonucleotides were prepared on a synthesizer (Applied
Biosystems, 392 DNA/RNA Synthesizer) utilizing chemicals
obtained from J.T. Baker and Applied Biosystems. The synthesis
began with a dC bonded to the solid phase (polystyrene, Applied
Biosystems, LV 200), in the trityl-off mode. After completion
of synthesis, the solid phase was dried under vacuum. The
oligonucleotide was cleaved from the solid support by treatment
with ammonia (33%) at 55 ◦C overnight and the solvent was
removed under vacuum. The residue was taken up in ultrapure
water and separated by filtration from the solid phase and
subsequently purified by preparative PAGE. The identity of the
oligonucleotides was verified by ESI mass spectrometry.
9 (a) W. K. Yang, J. D. Fabbri, T. M. Willey, J. R. I. Lee, J. E. Dahl,
R. M. K. Carlson, P. R. Schreiner, A. A. Fokin, B. A. Tkachenko, N.
A. Fokina, W. Meevasana, N. Mannella, K. Tanaka, X. J. Zhou, T.
van Buuren, M. A. Kelly, Z. Hussain, N. A. Melosh and Z.-X. Shen,
Science, 2007, 316, 1460; (b) S. Roth, D. Leuenberger, J. Osterwalder,
J. E. P. Dahl, R. M. K. Carlson, B. A. Tkachenko, A. A. Fokin, P. R.
Schreiner and M. Hengsberger, Chem. Phys. Lett., 2010, 495, 102.
10 (a) L. Wanka, C. Cabrele, M. Vanejews and P. R. Schreiner, Eur. J.
Org. Chem., 2007, 1474; (b) A. A. Fokin, A. Merz, N. A. Fokina, H.
Schwertfeger, S. G. Liu, J. E. P. Dahl, R. M. K. Carlson and P. R.
Schreiner, Synthesis, 2009, 909.
Circular dichroism (CD)-measurements
11 A. Baccaro and A. Marx, Chem.–Eur. J., 2010, 16, 218.
CD spectra were measured on a Jasco J-715 instrument in
a phosphate-buffer (20 mM K2HPO4/KH2PO4, pH = 7.0, 1.0
M NaCl) at 25 ◦C. Before the CD measurements, a heating
step to 95 ◦C for 5 min followed by slowly cooling to 4 ◦C
was performed to DNA duplex formation. For background
subtraction a spectrum of the phosphate-buffer was separately
measured. The scanning speed was 20 nm/min. The spectrum
results from the sum of 10 measurements subtracting the spectrum
of the phosphate-buffer.
ˇ
12 P. Capek, H. Cahova´, R. Pohl, M. Hocek, C. Gloeckner and A. Marx,
Chem.–Eur. J., 2007, 13, 6196.
13 S. Ja¨ger, G. Rasched, H. Kornreich-Leshem, M. Engeser, O. Thum and
M. Famulok, J. Am. Chem. Soc., 2005, 127, 15071.
14 T. Ohbayashi, M. Kuwahara, M. Hasegawa, T. Kasamatsu, T. Tamura
and H. Sawai, Org. Biomol. Chem., 2005, 3, 2463.
15 S. H. Weisbrod and A. Marx, Chem. Commun., 2007, 1828.
16 A. Baccaro, S. H. Weisbrod and A. Marx, Synthesis, 2007, 13,
1949.
17 S. Obeid, M. Yulikov, G. Jeschke and A. Marx, Angew. Chem., 2008,
120, 6886; S. Obeid, M. Yulikov, G. Jeschke and A. Marx, Angew.
Chem., Int. Ed., 2008, 47, 6782.
Melting temperature (Tm)-measurements
18 H. Sawai, A. N. Ozaki, F. Satoh, T. Ohbayashi, M. M. Masud and H.
Ozaki, Chem. Commun., 2001, 2604.
Melting curves were recorded on Varian Cary 100 bio UV/Vis
ˇ
19 S. Ikonen, H. Mac´ıcˇkova´-Cahova´, R. Pohl, M. Sanda and M. Hocek,
Spectrophotometer. 10 mM of the sample (same as for CD spectra)
Org. Biomol. Chem., 2010, 8, 1194.
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 7482–7490 | 7489
©

View Article Online
20 M. Hocek and M. Fojta, Org. Biomol. Chem., 2008, 6, 2233.
21 G. M. Dubowchik, L. Padilla, K. Edinger and R. A. Firestone, J. Org.
Chem., 1996, 61, 4676.
22 T. Courtney, D. E. Johnston, M. A. McKervey and J. J. Rooney, J.
Chem. Soc., Perkin Trans. 1, 1972, 2691.
23 H. Duddeck, F. Hollowood, A. Karim and M. A. McKervey, J. Chem.
Soc., Perkin Trans. 2, 1979, 360.
35 N. D. Sinha, J. Biernat, J. McManus and H. Ko¨ster, Nucleic Acids Res.,
1984, 12, 4539.
36 M. Chandra, S. Keller, Y. Luo and A. Marx, Tetrahedron, 2007, 63,
8576.
37 C. Gloeckner, K. B. M. Sauter and A. Marx, Angew. Chem., 2007, 119,
3175; C. Gloeckner, K. B. M. Sauter and A. Marx, Angew. Chem., Int.
Ed., 2007, 46, 3115.
24 Z.-B. Li, Z.-H. Wu, K. Chen, F. T. Chin and X.-Y. Chen, Bioconjugate
Chem., 2007, 18, 1987.
38 M. J. Gait, An introduction to modern methods of DNA synthesis. In
Oligonucleotide Synthesis: A practical approach., IRL Press, Oxford,
1984.
25 G. Lu, S. Lam and K. Burgess, Chem. Commun., 2006, 1652.
26 M. J. Robins and P. J. Barr, Tetrahedron Lett., 1981, 22, 421.
27 K. Sonogashira, Y. Tohda and N. Hagihara, Tetrahedron Lett., 1975,
16, 4467.
39 F. Eckstein, Oligonucleotide and analogues: A practical approach, IRL
Press, Oxford, 1991.
40 I. Detmer, D. Summerer and A Marx, Chem. Commun., 2002, 2314.
41 S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett., 1981, 22, 1859.
42 S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223.
43 S. L. Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123.
44 L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24, 245.
45 M. H. Caruthers, Science, 1985, 230, 281.
46 M. H. Caruthers, A. D. Barone, S. L. Beaucage, D. R. Dodds, E.
F. Fisher, L. J. McBride, M. Matteucci, Z. Stabinsky and J.-Y. Tang,
Methods Enzymol., 1987, 154, 287.
28 J. D. Kahl and M. M. Greenberg, J. Am. Chem. Soc., 1999, 121, 597.
¨
29 T. Kova´cs and L. Otvo¨s, Tetrahedron Lett., 1988, 29, 4525.
30 M. Yoshikava, T. Kato and T. Takenish, Tetrahedron Lett., 1967, 8,
5065.
31 J. Ludwig, Biochim. Biophys. Acad. Sci. Hung., 1981, 16, 131.
32 A. Rospigliosi, R. Ehlich, H. Hoerber, A. Middelberg and G. Mog-
gridge, Langmuir, 2007, 23, 8264.
33 R. H. E. Hudson and A. Ghorbani-Choghamarani, Synlett, 2007, 870.
34 N. Usman, K. K. Ogilvie, M.-Y. Jiang and R. J. Cedergrens, J. Am.
Chem. Soc., 1987, 109, 7845.
47 O. Dahl, J. Nielson and B. H. Dahl, Nucleic Acids Res., 1987, 15,
1729.
7490 | Org. Biomol. Chem., 2011, 9, 7482–7490
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The Royal Society of Chemistry 2011
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