Towards the synthesis of inosine building blocks for the preparation of oligonucleotides with hydrophobic alkyl chains between the nucleotide units

Article abstract of DOI:10.3390/molecules14114326

The scientific objective of the research reported in this manuscript was the synthesis of novel phosphoramidite building blocks for the preparation of lipophilic oligonucleotides. Reaction of inosine (4) with 4-oxopentyl-4- methylbenzoate (2c) in the pres

Full text of DOI:10.3390/molecules14114326

Molecules 2009, 14, 4326-4336; doi:10.3390/molecules14114326
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Towards the Synthesis of Inosine Building Blocks for the
Preparation of Oligonucleotides with Hydrophobic Alkyl Chains
Between the Nucleotide Units †
Karl Köstler and Helmut Rosemeyer *
Organische Materialchemie und Bioorganische Chemie, Institut für Chemie, Fachbereich
Biologie/Chemie, Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück, Germany
†
Dedicated to Prof. Dr. Frank Seela on the occasion of his 70th birthday.
* Author to whom correspondence should be addressed; E-Mail: Helmut.Rosemeyer@uos.de.
Received: 18 September 2009; in revised form: 19 October 2009 / Accepted: 23 October 2009 /
Published: 26 October 2009
Abstract: The scientific objective of the research reported in this manuscript was the
synthesis of novel phosphoramidite building blocks for the preparation of lipophilic
oligonucleotides. Reaction of inosine (4) with 4-oxopentyl-4-methylbenzoate (2c) in the
presence of triethyl orthoformate and 4M HCl in 1,4-dioxane gave a diastereoisomeric
mixture of the ketals 5. Subsequent 4,4’-dimethoxytritylation at the 5’-hydroxyl afforded
(R)-6 + (S)-6 which could be separated chromatographically. Detoluoylation gave
compounds (R)-7 and (S)-7. Phosphitylation of a diastereoisomeric mixture of 7 led to a
mixture of four diastereoisomers of the corresponding 2-cyanoethylphosphoramidites 8.
Keywords: lipophilic oligonucleotides; phosphoramidites; nucleoside ketals
Introduction
The low cell membrane permeability of oligonucleotides is one major drawback of antisense gene
therapy. Therefore, as early as the eighties antisense and antigene oligomers were prepared which
carried lipophilic groups, mostly at the termini. Other lipophilic modifications comprise derivatization
of: (i) the nucleobases, (ii) of the glyconic residues or (iii) of the phosphodiester moiety of the
oligonucleotides with hydrophobic side groups [1]. Of particular importance for the stability and
activity of antisense oligonucleotides proved to be their modification with terminal cholesterol residues

Molecules 2009, 14
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[2] which culminated in the development of the so-called “antagomirs” which are constructed from
oligo(2’-OMe-ribonucleotides) carrying several phosphorothioate linkages at both termini and one
cholesterol residue at the 5’-end. Such therapeutics have been successfully used for the in-vivo
silencing of microRNAs [3]. The various applications of “nucleolipids” (nucleoside, nucleotide and
oligonucleotide-based amphiphiles) were reviewed recently [4]. Appending a cholesterol moiety to an
oligonucleotide, however, renders it so hydrophobic that its solubility in aqueous solvents is
significantly reduced and its handling becomes problematic [5]. For example, a trimer consisting of
three wobble nucleotides 2’-deoxyinosine [5’-d(IpIpI)] exhibits a calculated logP value of -6.67 ± 1.06
while its 5’-cholesterol derivative shown in Figure 1 exhibits a logP of +1.79 ± 1.27.
Figure 1. 3D-Optimized structure of a 5’-cholesterol labelled trimer [5’-Chol-5’d(IpIpI)]
using ChemSketch, 3D viewer, version 12.0 (Advanced Chemistry Developments, Inc.
Toronto, Canada, http://www.acdlabs.com).
We, therefore, designed an oligonucleotide building block on the basis of an O-2’,3’-cyclic ketal –
first with hypoxanthine as wobble base – which allows a stepwise hydrophobization of an
oligonucleotide and which can be incorporated into all positions of a growing nucleic acid chain by
conventional solid-phase synthesis yielding a general structure shown in Figure 2. In this manuscript
we report first steps toward the preparation of this new kind of oligonucleotide building block.

Molecules 2009, 14
Figure 2. Left: General structure of an oligonucleotide with a lipophilic internucleotide
4328
linkage. Right: 3D-Optimized structure of a trimer [5’-d(IpI*pI)] with an inosine O-2’,3’-
ketal with three methylene groups in the central position of the oligomer.
Results and Discussion
In order to get an impression about the hydrophobization effect of the incorporation of one or more
O-2’,3’-cyclic ketal derivative of inosine , I*, (with three CH2 groups in the ketal side chain) into an
oligonucleotide, we first calculated the logP values [6] of a series of inosine trimers with varying
substitutions of inosine by inosine ketals (I*) with a chain length of three methylene groups. Figure 3
and Table 1 display the increase of lipophilicity with the increasing number of modifications as well as
the influence of the position of incorporation.
It can be seen that in case of incorporation of either one or two modified units into an inosine trimer
the resulting logP value depends on the position of incorporation: Substitution of one inosine by an
inosine ketal at the 3’-end results in an oligomer (entry 2, Table 1) with a free primary hydroxyl group
at the 5’-terminus; this oligomer shows a significantly lower lipophilicity than the corresponding
trimers carrying the modified inosine derivative either in the middle position (entry 3) or at the 5’-end
(entry 4). An analogous result can be seen for trimers carrying two modifications (entries 5-7, Table
1). The synthesis of 2’-cyanoethyl phosphoramidite building blocks of inosine O-2’,3’-cyclic ketals is
shown in Scheme 1. First, we converted the (ω-1) ketoalcohols 1a-c to the tolyl-protected compounds
2a-c. Exemplary, Figure 4 displays the three-dimensional structure of 2b obtained from an X-ray
analysis [7].
Figure 3. Calculated logP values of inosine oligonucleotide trimers as a function of the
number of inosine O-2’,3’-cyclic ketal derivatives (see Figure 2).
1
0
-1
-2
-3
-4
-5
-6
-7
-8
0
1
2
3
n x I*

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Table 1. Calculated logP values of inosine oligonucleotide trimers as a function of the
number and position of inosine O-2’,3’-cyclic ketal derivatives (see Figure 2).
Number of
modified units
Calculated
logP Values
-6.67 ± 1.06
-5.29 ± 1.12
-4.29 ± 1.16
-4.29 ± 1.16
-2.92 ± 1.22
-2.92 ± 1.22
-1.91 ± 1.29
-0.54 ± 1.32
Entry
Compound
1
2
3
4
5
6
7
8
5’-d(IpIpI)
5’-d(IpIpI*)
5’-d(IpI*pI)
5’-d(I*pIpI)
5’-d(IpI*pI*)
5’-d(I*pIpI*)
5’-d(I*pI*pI)
5’-(I*pI*pI*)
0
1
1
1
2
2
2
3
Earlier, it had been shown that reaction of inosine (4) with (ω-1) ketoesters such as ethyl levulinate
or unsymmetrical ketones such as pentan-2-one leads to O-2’,3’-ketals with predominant or even
exclusive formation of the (R) configuration at the newly formed stereogenic center [8-12] [the (R)-
and (S)-notation within this manuscript refers always to the configuration at the stereogenic center of
the ketal moiety]. We now prepared the keto esters 2a-c, of which only compound 2c had been
described earlier [13]. All attempts to react inosine with compound 2a in the presence of triethyl
orthoformate and 4M HCl in 1,4-dioxane in various solvents failed. Next, we, therefore, converted
compound 2a into the open acetal 3. A subsequent transacetalisation with inosine (4) which usually
occurs under mild reaction conditions also failed. Also ketalisation of inosine with the elongated (ω-1)
ketoester 2b did not show the desired result. The reason for this result might be an electrostatic
repulsion of the ester oxygen atom and the ribose oxygen.
Figure 4. Ball and stick model of compound 2b (with the exception of the hydrogen atoms,
which are represented by use of spheres with a common isotropic radius, all other atoms
are represented as thermal displacement ellipsoids showing 50% of the probability of the
corresponding atom.

Molecules 2009, 14
4330
Scheme 1. Synthesis of the target structures. In case of the ketals (5–8) the R
diastereoisomers always are shown.

Molecules 2009, 14
4331
At least, reaction of 4 with 4-oxopentyl-4-methylbenzoate, (2c) under the reaction conditions
1
13
mentioned above gave the desired product 5, however, H- and C-NMR spectroscopy proved the
formation of a diastereoisomeric mixture [(R)-5 + (S)-5]. Integration of the 1H-NMR resonances of the
clearly separated ketal Me groups indicated a ratio of 47% of the (R)- and 53% of the (S)-
diastereoisomer. The assignment of the NMR resonances of the different methyl groups as well as of
other signals was made on the basis of a comparison with the 1H- and 13C-NMR spectra of (R)-2’,3’-O-
(3-carboxy-1-methylpropyliden)adenosine from which an X-ray analysis had been performed earlier
[14] as well as by gradient-selected homo- and heteronuclear correlation spectroscopy.
A chromatographic separation of the mixture (R)-5 + (S)-5 proved extremely difficult; only a TLC
with a 20-fold development in EtOAc/toluene (97:3, v/v) showed the presence of two products.
Because the positioning of the ketal side chain is of decisive influence on the topology of
oligonucleotides carrying such modified building blocks, a separation of the diastereoisomers is at
least inevitable. Next, we converted the mixture (R)-5 + (S)-5 into the 4,4’-dimethoxytriphenylmethyl
derivatives (R)-6 + (S)-6. On this stage the diastereoisomeric mixture could be separated silica gel
chromatographically by elution with EtOAc/toluene (97:3, v/v). Both diastereoisomers were
characterized by 1H-NMR spectra.
In order to prove if a chromatographic separation is also possible on the stage of the de-toluoylated
compounds a mixture of (R)-6 + (S)-6 was deprotected by treatment with conc. aq. ammonia/MeOH
(1:1, v/v). After 72 h a mixture of (R)-7 + (S)-7 was isolated in moderate 38% yield. As it was found
that a chromatographic separation of the diastereoisomers was very difficult, the separation was
performed on the stage of compounds 6. De-toluoylation was performed on either the stage of a
diastereoisomeric mixture or on the stage of the separated isomers and afforded (R)-7 and (S)-7. A first
subsequent phosphitylation [15] of a mixture of (R)-7 + (S)-7 with chloro-(2-cyanoethoxy)-N,N-
diisopropylethylaminophosphane (CH2Cl2, Hünig’s base, 3 h) gave, after chromatography, a mixture
of four diastereoisomers (additional RP and SP diastereoisomers) in 67% total yield. The
phosphitylation of the separated (R)-7 and (S)-7 isomers, their incorporation into oligonucleotides as
well as the base pairing properties of such oligomers will be published in following manuscripts. We
anticipate that appending of the novel building blocks to one or both termini of an unmodified
oligonucleotide will lead to gap-mers with unaltered binding to their complementary strands but that
their incorporation into the innermost part of a nucleic acid will give new and autonomous nucleic acid
pairing systems.
Experimental
General
Thin-layer chromatography (TLC): Silica gel 60 F254 plates (VWR, Darmstadt, Germany). UV-
Spectroscopy: U-3200 spectrophotometer (Hitachi, Japan); λmax in nm; ε in M-1cm-1. NMR Spectra
were recorded on AC-250 and AMX-500 spectrometers (Bruker BioSpin, Rheinstetten, Germany).
1
13
Operational frequencies: H-NMR: 250.13, 500.14 MHz; C-NMR: 62.896, 125.700 MHz. Chemical
shifts (δ values) are in parts per million relative to tetramethylsilane as internal standard.
Microanalyses were performed by Mikroanalytisches Labor Beller-Matthies (Göttingen, Germany).
Melting points were measured on a Büchi SMP 20 apparatus and are not corrected. The 3D-optimized

Molecules 2009, 14
4332
structures were obtained using the program ChemSketch/3D Viewer, version 12.0, from Advanced
Chemistry Development Inc., Toronto, Canada (http://www.acdlabs.com).
2-Oxopropyl-4-methylbenzoate (2a)
3-Hydroxopropan-2-one (1a, 1.13 g, 15 mmol) was dissolved in anhydr. pyridine (3 mL) and
cooled in an ice bath. After drop-wise addition of tolyl chloride (1.55 g, 10 mmol) the mixture was
stirred for 30 min, warmed up to ambient temperature and stirred overnight. Then, the reaction mixture
was poured into ice-water (100 mL), acidified by addition of conc. hydrochloric acid and warmed up
to ambient temperature. After extraction with CHCl3 the organic layer was separated and washed with
5% aqueous NaHCO3. The organic phase was dried (Na2SO4) and evaporated. After drying under high
vacuum 1.7 g (88%) of the title compound were obtained as colourless crystals. M.p. 48 °C; TLC
(silica gel, EtOAc/n-hexane, 33:67, v/v): Rf, 0.7; Anal. calcd. for C11H12O3 (192.211): C, 68.74; H,
1
6.29. Found: C, 68.65; H, 6.18; H-NMR (d6-DMSO): δ, 2.16 (3H, s, CH3-arom.); 2.40 (3H, s, CH3-
13
C=O); 4.99 (2H, s, CH2); 7.35, 7.89 (2 × 2H, d, J = 7.5 Hz, H-arom.); C-NMR (d6-DMSO): δ, 21.6
(CH3-arom.); 26.4 (CH3-C=O); 69.1 (CH2); 126.9, 129.80, 144.5 (4 C-arom.); 165.6 (C=O-ester);
202.3 [C(=O)-CH3].
2,2-Diethoxypropyl-4-methylbenzoate (3)
To compound 2a (0.96 g, 5 mmol) was added triethyl orthoformate (0.82 g, 5.5 mmol), anhydrous
ethanol (0.23 g, 5 mmol) and one drop of conc. sulphuric acid. After stirring at room temperature
overnight the mixture was evaporated and chromatographed on silica gel 60 (6 × 10 cm, EtOAc/n-
hexane, 33:67, v/v). From the main fraction the title compound was obtained as a colourless foam
1
upon evaporation. Yield: 0.43 g (32%). TLC (silica gel, EtOAc/n-hexane, 33:67, v/v): Rf, 0.95; H-
NMR (d6-DMSO): δ, 1.10 (3H, t, J = 7.5 Hz, CH3-ethyl); 1.36 [CH3-C(OEt)2]; 2.50 (3H, s, CH3-
arom.); 3.47 (2H, q, J = 7.5 Hz, CH2-ethyl); 4.22 (2H, s, CH2); 7.34, 7.88 (2 × 2H, d, J = 7.5 Hz, H-
13
arom.); C-NMR (d6-DMSO): δ, 15.8 (2 CH3CH2O); 21.6 (2 CH3); 56.1 (2 CH3CH2O); 65.3 (CH2);
127.2, 129.8, 144.3 (4 C-arom.); 99.4 (C-ketal); 165.7 (C=O-ester).
3-Oxobutyl-4-methylbenzoate (2b)
4-Hydroxybutan-2-one (1b, 1.32 g, 15 mmol) was reacted with tolyl chloride (1.55 g, 10 mmol) and
worked up as described for 2a. After crystallization from hot propan-2-ol colourless crystals of 2b
(1.55 g, 75%) were obtained. M.p. 37 °C; TLC (silica gel, EtOAc/n-hexane, 33:67, v/v): Rf, 0.7; Anal.
1
calcd. for C12H14O3 (206.238): C, 69.88; H, 6.84. Found: C, 69.74; H, 6.65; H-NMR (d6-DMSO): δ,
2.10 (3H, s, CH3-arom.); 2.32 (3H, s, CH3-C=O); 2.87 (2H, t, J = 5.5 Hz, CH2-C=O); 4.36 (2H, t,
13
J = 7.5 Hz, CH2-O); 7.25, 7.74 (2 2H, d, J = 7.3 Hz, H-arom.); C-NMR (d6-DMSO): δ, 21.6 (CH3-
arom.); 30.4 (CH3-C=O); 42.1 (CH2-C=O); 60.2 (CH2-O); 127.4, 129.7, 144.1 (4 C-arom.); 166.1
(C=O-ester); 208.2 [C(=O)-CH3].

Molecules 2009, 14
4333
4-Oxopentyl 4-methylbenzoate (2c) [13]
5-Hydroxypentan-2-one (1c, 1.53 g, 15 mmol) was reacted with tolyl chloride (1.55 g, 10 mmol)
and worked up as described for 2a. After column chromatography (silica gel 60H, 6 × 10 cm,
EtOAc/n-hexane, 33:67, v/v) the main fraction was pooled and evaporated to dryness to give a
colourless oil of 2c (1.72 g, 78%). TLC (silica gel, EtOAc/n-hexane, 33:67, v/v): Rf, 0.68; Anal. calcd.
1
for C13H16O3 (220.264): C, 70.89, H, 7.32. Found: C, 70.78; H, 7.21; H-NMR (d6-DMSO): δ, 1.89
(2H, pq, J = 7.5 Hz, CH2); 2.10 (3H, s, CH3-C=O); 2.38 (3H, s, CH3-arom.); 2.60 (2H, t, J = 7.5 Hz,
13
CH2-C=O); 4.21 (2H, t, J = 7.5 Hz, CH2-O); 7.32, 7.84 (2 × 2H, d, J = 7.5 Hz, H-arom.); C-NMR
(d6-DMSO): δ, 21.6 (CH2); 23.0 (CH3-arom.); 30.2 [CH3-C(=O)]; 40.3 [CH2-C(=O)]; 64.4 (CH2-O);
127.5, 129.7, 144.0 (4 C-arom.); 166.2 (C=O-ester); 208.2 [C(=O)-CH3].
3-((3aS,4R,6R,6aS)-4-(hydroxymethyl)-2-methyl-6-(6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuro[3,4-
d][1,3]dioxol-2-yl)propyl-4-methylbenzoate, diastereoisomeric mixture [(R)-5 + (S)-5]
Anhydrous inosine (4, 0.54 g, 2 mmol) was suspended in anhydrous MeCN (7.5 mL), and
compound 2c (2.2 g, 10 mmol) and triethyl orthoformate (0.5 ml, 3 mmol) were added. Upon drop-
wise addition of 4M HCl in 1,4-dioxane (1.75 mL) the inosine went into solution. The reaction mixture
was stirred at ambient temperature for 24 h and then evaporated to dryness. Column chromatography
(silica gel 60, 6 × 15 cm, stepwise elution: (i) CHCl3/MeOH, 97:3, 250 ml; (ii) CHCl3/MeOH, 9:1, 250
ml, v/v, each) afforded one main fraction from which a diastereoisomeric mixture of compound 5 (0.56
g, 60%) was obtained after evaporation of the solvent as a colourless solid. M.p. 229 °C (decomp.);
TLC (silica gel, CHCl3/MeOH, 9:1, v/v): Rf, 0.62; UV (MeOH): λmax, 248 nm (ε = 28,200 M-1cm-1;
ε260 = 18,200 M-1cm-1); Anal. calcd. for C23H26N4O7 (470.514): C, 58.72%; H, 5.57%; N, 11.91%.
1
Found: C, 58.80%, H, 5.49%; N, 11.81%; H-NMR (d6-DMSO): δ, 12.5 (1H, br., s, NH); 8.3002 and
8.2970 (2 × 1H, 2 s, H-2); 8.0731 and 8.0668 (2 × 1H, 2 s, H-8); 7.86 and 7.83 (2 × 2H, 2 d, J = 5.0
Hz, H-arom.), 7.34 and 7.30 (2 × 2H, 2 d, J = 5.0 Hz, H-arom.); 6.14 (2 × 1H, 2 d, J(1’,2’) = 2.5 Hz,
H-1’); 5.341-5.359 and 5.304-5.286 (2 × 1H, 2 × dd, J(2’,1’) = 2.5 Hz, J(2’,3’) = 6.5 Hz, H-2’); 5.10
(1H, m, 5’-OH); 4.994-4.956 (2 × 1H, 2 × dd, J(3’,2’) = 6.5 Hz), J(3’,4’) = 2.5 Hz, H-3’); 4.33 (1H, m,
H-4’); 4.255-4.235 (2H, m, CH2-O-C(O)); 3.52 (2H, m, H2-5’); 2.39 and 2.38 (2 × 3H, 2 × s, CH3-
arom.); 1.93 (2H, m, CH2); 1.75 (2H, m, CH2); 1.538 (3H, s, CH3-ketal (R)); 1.333 (3H, s, CH3-ketal
13
(S)); C-NMR (d6-DMSO): δ, 146.5 (C-2); 148.2 (C-4); 115.2 (C-5); 157.0 (C-6); 144.0 (C-8); 87.4
(C-1’); 84.3 and 84.7 (C-2’); 81.7 and 82.1 (C-3’); 90.0 (C-4’); 61.9 (C-5’); 23.2 and 23.9 (CH3-ketal);
114.8 (C-quart. ketal); 35.0 and 35.6 (CH2); 21.6 (CH2); 64.8 (CH2-O); 166.2 (C(=O)-ester); 124.9,
129.6, 127.6 and 139.2 (C-arom.); 25.5 (CH3-arom.).
3-((3aS,4R,6R,6aS)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-2-methyl-6-(6-oxo-1,6-dihydro-
purin-9-yl)-tetrahydrofuro[3,4-d][1,3]dioxol-2-yl)propyl 4-methylbenzoate, diastereoisomeric mixture
[(R)-6 + (S)-6]
The diastereoisomeric mixture (R)-5 + (S)-5 (470 mg, 1.0 mmol) was dried by repeated co-
evaporation from anhydrous pyridine. The residue was dissolved in anhydrous pyridine (6 mL), 4,4’-
dimethoxytriphenylmethyl chloride (643 mg, 1.9 mmol) was added, and the reaction mixture was

Molecules 2009, 14
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stirred for 3 h under a N2 atmosphere. Then, 5% aq. NaHCO3 (30 ml) was added, and the mixture was
extracted three times with CHCl3 (30 mL, each). The combined organic layers were dried (Na2SO4)
and evaporated to dryness (high vacuum). Column chromatography (silica gel 60, 6 × 20 cm,
CHCl3/MeOH, 97:3, v/v) afforded one main fraction from which the title compound was obtained as
colourless glassy foam (0.41 g, 53%). TLC (silica gel, CHCl3/MeOH, 97:3, v/v): Rf, 0.38; UV
(MeOH): λmax, 242 nm (ε = 47,300 M-1cm-1; ε260 = 26,000 M-1cm-1); Anal. calcd. for C44H44N4O9
(772.842): C, 68.38%; H, 5.74%; N, 7.25%. Found: C, 68.45; H, 5.84; N, 7.09. The diastereoisomeric
mixture [(R)-6 + (S)-6] was separated by column chromatography (silica gel 60, 6 × 25 cm,
EtOAc/toluene, 98:2, v/v).
3-((2R,3aS,4R,6R,6aS)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-2-methyl-6-(6-oxo-1,6-
dihydropurin-9-yl)-tetrahydrofuro[3,4-d][1,3]dioxol-2-yl)propyl 4-methylbenzoate [(R)-6]
From the slower migrating zone the (R) diastereoisomer was isolated as colourless foam. TLC
(silica gel, EtOAc/toluene, 98:2, v/v): Rf, 0.45; 1H-NMR (d6-DMSO): δ, 13.23 (1H, s, NH); 8.16 (1H,
s, H-2); 7.94 (1H, s, H-8); 8.00 (2 × 1H, d, J = 7.5 Hz, DMTr); 7.40-7.19 (13 H, m, DMT); 6.81 (2 ×
2H, H-arom.); 6.17 (1H, d, J(1’,2’) = 2.5 Hz, H-1’); 5.46 (1H, dd, J(2’,1’) = 2.5 Hz, J(2’,3’) = 5.0 Hz,
H-2’); 5.00 (1H, dd, J(3’,2’) = 5.0 Hz, J(3’,4’) = 3.0 Hz, H-3’); 4.61 (1H, m, H-4’); 4.44 (2H, m, H2-
5’); 3.78 (6H, s, 2 × OCH3); 3.33 (2H, m, CH2); 2.44 (3H, s, CH3-arom.); 1.43 (2H, m, CH2); 1.30 (3H,
s, CH3-ketal).
3-((2S,3aS,4R,6R,6aS)-4-(bis((4-methoxyphenyl)(phenyl)methoxy)-methyl)-2-methyl-6-(6-oxo-1,6-
dihydropurin-9-yl)-tetrahydrofuro[3,4-d][1,3]dioxol-2-yl)propyl 4-methylbenzoate [(S)-6]
From the faster migrating zone the (S) diastereoisomer was isolated as colourless foam. TLC (silica
1
gel, EtOAc/toluene, 98:2, v/v): Rf, 0.55); H-NMR (d6-DMSO): δ, 12.83 (1H, s, NH); 7.97 (1H, s, H-
2); 7.90 (1H, s, H-8); 7.36-7.22 (13H, m, DMT); 6.80-6.77 (4H, m, H-arom.); 6.16 (1H, m, H-1’); 5.37
(1H, d, J(2’,3’) = 7.5 Hz, H-2’); 4.99 (1H, d, J(3’,2’) = 7.5 Hz, H-3’); 4.57 (1H, m, H-4’); 4.35 (2H, m,
H2-5’); 3.78 (6H, s, 2 × OCH3); 3.33 (2H, m, CH2); 2.41 (3H, m, CH3-arom.); 1.85 (2H, m, CH2); 1.65
(3H, s, CH3-acetal); 1.29 (2H, m, CH2).
3-((3aS,4R,6R,6aS)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(3-hydroxypropyl)-2-methyl-
tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1H-purin-6(9H)-one, diastereoisomeric mixture [(R)-7 + (S)-7]
A diastereoisomeric mixture of (R)-6 + (S)-6 (772 mg, 1.0 mmol) was dissolved in 7M NH3 in
MeOH (20 mL) and stirred for 72 h at ambient temperature. After evaporation of the solvent the raw
product was chromatographed (silica gel 60, (i) CHCl3/MeOH, 97:3, (ii) CHCl3/MeOH, 9:1, v/v).
From the main fraction the title compound [(R)-7 + (S)-7] was obtained as colourless solid (0.25 g,
38%). TLC (silica gel, CHCl3/MeOH, 9:1, v/v): Rf, 0.62; UV (MeOH): λmax, 240 nm (ε = 38,100
M-1cm-1; ε260 = 23,000 M-1cm-1); Anal. calcd. for C36H38N4O8 (654.709): C, 66.04%; H, 5.85%; N,
8.56%. Found: C, 66.09%; H, 5.91%; N, 8.41%. The diastereoisomeric mixture [(R)-7 + (S)-7] proved
to be chromatographically separable only with difficulties; therefore, the precursor diastereoisomers
(R)-6 and (S)-6 were de-toluoylated separately and worked up as described above.

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(R)-7: H-NMR (d6-DMSO): δ, 12.34 (s, br., 1H, NH); 8.22 (s, 1H, H-2); 7.93 (s, 1H, H-8); 7.286-
6.763 (4m, 13H, H-aromat.); 6.20 (d, 1H, J(1’,2’) = 1.0 Hz), H-1’); 5.39 (dd, 1H, J(2’,1’) = 1.0 Hz,
J(2’,3’) = 6.5 Hz), H-2’); 4.91 (dd, 1H, J(3’,2’) = 6.5 Hz, J(3’,4’) = 3.0 Hz, H-3’); 4.50 (1, 1H, J = 5.5
Hz, OH); 4.32 (pq, 1H, J(4’,5’) = 3.0 Hz, H-4’); 3.73, 3.72 (2s, 2 × 3H, 2 OCH3); ~3.4 (m, CH2); 3.22,
3.04 (2m, 2H, H2-5’); 1.78 (m, 2H, CH2); 1.61 (m, 2H, CH2); 1.27 (s, 3H, CH3-ketal).
(S)-7: 12.4 (s, br. 1H, NH); 8.24 (s, 1H, H-2); 7.95 (s, 1H, H-8); 7.287-6.762 (4m, 13H, H-aromat.);
6.25 (d, 1H, H-1’); 5.34 (d, 1H, J(2’,1’) = 5.0 Hz, H-2’); 4.92 (m, 1H, H-3’); 4.50 (m, 1H, OH); 4.35
(m, 1H, H-4’); 3.45 (m, 2H, CH2); 3.724, 3.723 (2s, 2 × 3H, 2 OCH3); 3.23, 3.07 (2 m, 2H, H2-5’);
1.59 (m, 2H, CH2); 1.44 (m, 2H, CH2); 1.49 (s, 3H, CH3-ketal).
3-((3aS,4R,6R,6aS)-4-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-2-methyl-6-(6-oxo-1,6-
dihydropurin-9-yl)-tetrahydrofuro[3,4-d][1,3]dioxol-2-yl)propyl 2-cyanoethyldiisopropyl-
phosphoramidite, diastereoisomeric mixture [(R)-8 + (S)-8]
The diastereoisomeric mixture (R)-7 + (S)-7 (115 mg, 0.175 mmol) was suspended in abs. CH2Cl2
(7 mL) and N-ethyldiisopropylamine (345 µL, 1.97 mmol) as well as chloro-(2-cyanoethoxy)-N,N-
diisopropylethylaminophosphane (340 µL, 1.53 mmol) were added. After 3 h at ambient temperature
(N2 atmosphere). The reaction mixture was chromatographed (silica gel 60, 6 × 10 cm, CHCl3/MeOH,
9:1, v/v), and the content of the main fraction was pooled. Evaporation of the solvent gave the title
phosphoramidites as colourless foam (0.10 g, 67%). TLC (silica gel, CHCl3/MeOH, 9:1, v/v): Rf, 0.50,
0.54, 0.58, 0.61 (4 diastereoisomers); 31P-NMR (CDCl3): δ, 147.44, 147.36, 139.79, 139.74.
Conclusions
We have prepared the first phosphoramidite building blocks for automated nucleic acids synthesis
which can be incorporated into a growing nucleic acid chain at any position. The novel units
hydrophobize the DNA or RNA oligomers stepwise. Furthermore, using either the (R)- or the (S)-
diastereoisomer of such a novel building block opens the possibility to place a kink within the nucleic
acid at a predetermined position and to expose therewith a certain part of the oligomer.
Acknowledgements
We greatly acknowledge the excellent technical assistance by Marianne Gather (NMR spectra) and
Anja Schuster (elemental analyses). We are thankful to Prof. Dr. Uwe Beginn for excellent laboratory
facilities.
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Sample Availability: Samples of compound 2c for further ketalization reactions are available directly
from the corresponding author.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).