Synthesis of functionalised nucleosides for incorporation into nucleic acid-based serine protease mimics

Article abstract of DOI:10.3390/12010114

The synthesis of nucleosides modified with an extra imidazole, carboxyl and hydroxyl group is described. These nucleosides can be incorporated into an oligonucleotide duplex, thus generating a novel type of serine protease mimic.

Full text of DOI:10.3390/12010114

Molecules 2007, 12, 114-129
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Synthesis of Functionalised Nucleosides for Incorporation into
Nucleic Acid-Based Serine Protease Mimics
Mieke A. Catry and Annemieke Madder*
Ghent University, Department of Organic Chemistry, Laboratory for Organic and Biomimetic
Chemistry, Krijgslaan 281 (S4), B-9000 Gent, Belgium.
* Author to whom correspondence should be addressed. E-mail: Annemieke.Madder@UGent.be
Received: 21 December 2006; in revised form: 29 January 2007 / Accepted: 30 January 2007 /
Published: 31 January 2007
Abstract: The synthesis of nucleosides modified with an extra imidazole, carboxyl and
hydroxyl group is described. These nucleosides can be incorporated into an oligonucleotide
duplex, thus generating a novel type of serine protease mimic.
Keywords: Nucleoside synthesis, serine protease mimic, DNA scaffold, 2’ modification.
Introduction
Enzymes are of great importance in nature because of their ability to catalyse reactions in a
selective and efficient way. The development of artificial catalysts which mimic enzymatic activity has
always been a very challenging area of research. Among the different enzymes, proteases have been
intensively studied because of their unequalled capacity for hydrolysing amide and peptide bonds
under mild conditions. One of the most studied serine proteases is α-chymotrypsin, and X-ray
diffraction studies give a good insight into the tertiary structure of the enzyme [1-4]. The active site
contains three amino acids which are essential for catalysis of the cleavage of amide bonds (Figure 1).
The hydrolysis is realized by the unique collaboration between Histidine-57 (His-57), Serine-195 (Ser-
195) and Aspartate-102 (Asp-102) [5].

Molecules 2007, 12
Figure 1: The catalytic triad of α-chymotrypsin for peptide hydrolysis.
115
His57
O
O
Ser195
Asp102
H
N
N
H O
O
O
α-chymotrypsin
H2N-R'
+
R
NH-R'
R
OH
H2O
Intrigued by this performance, several groups have tried to construct model systems with
hydrolytic activity and different peptide based systems have been developed in this context [6-16]. We
must bear in mind that stable secondary structures such as helices only become important for rather
long sequences of polypeptides. Therefore, synthetic polymers, synzymes and modified cyclodextrins
have also been studied as possible catalytic systems [17-21]. Although a big variety of artificial
hydrolases has been synthesized, none have even approached the efficiency of α-chymotrypsin.
Two approaches have been used in our laboratory to develop potentially catalytic hydrolytic
systems. In a first approach, we developed non-peptidic organic molecules containing an array of
functional groups in a suitable geometry for possible hydrolytic activity [22]. In a second
combinatorial approach, a dipodal scaffold based on a cholic acid template was synthesized and two
independent peptide chains were attached, each containing one residue of the catalytic triad [23, 24].
In addition, a model based on a tripodal scaffold, with a rigid structure possessing three independent
functionalised peptide chains, was also synthesized [25].
We recently became interested in the development of a totally new class of model systems based
on nucleic acid building blocks. A major drawback in using oligonucleotides in the design of synthetic
catalysts is that they lack functional groups with such diversity and properties as proteins in order to
act as bases or Lewis acids. However, it is tempting to equip oligonucleotides with the functional tools
of peptide chains by building block modification. This should result in a powerful combination of both
predefined structural organization (duplex formation) and presence of catalytic entities.
More specifically, we envisaged the creation of an active site via duplex formation between two
complementary sugar modified oligonucleotide strands (Figure 2). The cleavage of the target peptide
bonds will occur via functionalities in the minor and major groove of the DNA duplex. Both strands of
the duplex will have several different functional group modifications. As for the catalytic groups,
imidazole, alcohol and carboxylate groups would be included in accordance with the functional groups
in the active site of serine proteases. Depending on the specific site of incorporation of the
functionalities in the nucleoside building blocks, different spatial positions within the duplex grooves
can be reached. In a first stage we have chosen to synthesize a series of functionalised nucleosides
starting from 2’-amino-2’-deoxyuridine.

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116
Figure 2: Creation of an active site.
NH2
N
N
O
O
N
O
O
HN
H2N
N
N
O
HN
OH
O
O
N
O-
O
P
O-
O
P
O
O
O
NH
N
N
O
N
NH2
N
N
O
H
N
O
HN
O
O
O
O
HN
OH
O
P
O-
O
O
P
O-
O
O
NH2
N
O
N
O
N
N
O
O
HN
N
H2N
Since DNA synthesis has become a reliable and relatively simple automated process, the synthesis
of modified ribo- and deoxyribooligonucleotides is a standard procedure [26]. After the synthesis of
suitably modified nucleosides, a wide variety of oligonucleotides can be synthesized in an automated
way.
Results and Discussion
Figure 3 shows the envisaged building blocks. Starting from 2’-amino-2’-deoxyuridine, prepared
according to literature procedures, three different functional groups would be attached via an amide
bond [27, 28].
Figure 3: 2’-Amido modified building blocks.
O
H
N
N
HN
O
N
5'
O
R=
HO
O
OH
2'
OH
NH
R
OH
O

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117
An important aspect when synthesizing functionalised oligonucleotides is the appropriate
protection of the extra functionalities on the corresponding nucleoside building blocks. Not only
should these protecting groups survive the DNA synthesis conditions, but they should also be stable
under all chemical conditions used for construction of the required suitably protected
phosphoramidites.
Modification of oligonucleotides with an imidazole functionality has been described before. To
circumvent known problems with imidazole protection during nucleoside and oligonucleotide
synthesis, precursor-based techniques employing post-synthetic functionalization have been
developed. The imidazole moiety is introduced after oligonucleotide synthesis either at the 5’-end or
internally, using a base-modified building block [29, 30]. Post-synthetic conjugation to a 2’-amino
building block has been reported, but only using a long linker [31].
We chose to introduce the imidazole functionality in the nucleoside and to incorporate it into
oligonucleotides. As for imidazole containing phosphoramidite building blocks, the imidazole moiety
has been mostly connected to the nucleoside via the heterocyclic base. No phosphoramidite building
blocks containing an imidazole moiety on C 2’, connected via an amide bond, have been previously
described.
The use of a 2,4-dinitrophenyl group (DNP), which has been reported as a protecting group for the
imidazole functionality of His in peptide chemistry and in stepwise solid phase synthesis of
nucleopeptides, was our first choice [32, 33]. However, the DNP protection did not survive conditions
used during purification of the final DMTr-protected nucleoside. Because it has further been proven
that the DNP group gives yellow contaminated products after deprotection, we decided to change the
protecting group into a tert-butoxycarbonyl (Boc) one [34], which is compatible with the reagents
used during nucleoside- and oligonucleotide synthesis [35-37]. In principle, removal of the Boc group
on a solid support can be accomplished with a 10% TFA solution after automated synthesis [37].
However, it has been shown that such a TFA treatment can lead to a certain amount of depurination of
the oligonucleotide [34, 38]. The research groups of Gait and Herdewijn both proved that removal of
the Boc group from imidazole during standard deprotection procedures with saturated methanolic
ammonia after oligonucleotide synthesis is possible [35, 39], therefore in our second attempt, using
urocanic acid (1), the imidazole moiety was Boc-protected using di-tert-butyldicarbonate as shown in
Scheme 1.
NIm-Boc-protected urocanic acid 2 was further coupled with the dimethoxytrityl protected 2’-
amino-2’-deoxyuridine 4 in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC)
and N-hydroxysuccinimide (NHS) to give 5 in 68% yield. Coupling of the Boc-protected urocanic acid
2 with unprotected 2’-amino-2’-deoxyuridine followed by dimethoxytritylation of this amide also gave
the desired compound 5, albeit in lower yield. The dimethoxytrityl protected amine 4 was synthesized
starting from the known 2’-azido-2’-deoxyuridine (3) [27, 28]. Selective dimethoxytritylation of the
5’-hydroxyl function, followed by reduction of the azide gave us the dimethoxytrityl protected amine 4
in good yield.
Since incorporation of unprotected imidazole building blocks into oligonucleotides has been
accomplished in some cases, we also directly coupled urocanic acid (1) to dimethoxytrityl protected
2’-amino-2’-deoxyuridine 4 [40, 41]. This was achieved using EDC and NHS, yielding the desired
compound 6 in only moderate yield [42, 43]. Alternative coupling procedures using N,N’-dicyclo-

Molecules 2007, 12
118
hexylcarbodiimide (DCC) and NHS, N,N’-diisopropylcarbodiimide (DIC) and polystyrene-bound
1-hydroxybenzotriazole (HOBt) or the pentafluorophenol ester of urocanic acid (1) were not effective.
Scheme 1: Synthesis of imidazole-modified nucleoside building blocks 5 and 6.
O
O
a
HO
HO
N
N
N
N
O
H
1
2
O
O
N
O
N
O
N
HN
HN
HN
O
O
O
HO
R
DMTrO
DMTrO
c
N
O
b
N
O
O
OH N3
OH NH2
OH NH
O
3
4
5 R= BOC
6 R= H
a) di-tert-butyl dicarbonate, DMF, rt, overnight (quantitative); b) i. DMTr-Cl, pyridine, rt, 3d (95%),
ii. Pd/C, H2, MeOH, rt, 4h (66%); c) R= BOC: 2, EDC, N-hydroxysuccinimide, DMF, rt, 7h (68%);
R= H: 1, EDC, N-hydroxysuccinimide, DMF, rt, overnight (45%).
Base and sugar modification of nucleosides with a carboxylic acid has been described and
protection of the carboxylate function proved to be necessary for oligonucleotide synthesis. As for our
envisaged carboxylic acid building block, shown in Scheme 2, a derivative similar to 11, only differing
in the carboxylic acid protecting group, has been synthesized before. However, this modified
nucleoside was incorporated only at the end of oligonucleotide sequences [44]. The carboxyl group
was protected as a benzyl ester, which can be selectively cleaved by hydrogenolysis. However, the use
of hydrogen gas leads to significant reduction of double bonds in the pyrimidine bases and the final
modified oligonucleotide was adsorbed on charcoal of the Pd catalyst, requiring a catalytic phase
transfer procedure using Pd nanoparticles.
Methyl- and ethyl esters have also been used as carboxylic acid protecting groups. Basic hydrolysis
of these esters is frequently plagued by low yields, long reaction times, the need for large excesses of
reagents, and the formation of by-products that are difficult to separate [45-47].
In our quest for a more straightforward alternative avoiding additional deprotection steps, our first
choice of carboxylate protecting group was the 9-fluorenylmethyl (Fm) group. This group is often used
as a protecting group for Asp in the stepwise solid-phase synthesis of nucleopeptides [48]. This
protecting group is stable under the conditions used in oligonucleotide synthesis and can be removed
with the standard oligonucleotide deprotection reagents. However, loss of the Fm group was observed
under conditions used for purification of the required DMTr-protected nucleoside.

Molecules 2007, 12
119
Since for other carboxylate-modified building blocks allyl protection was reported to be removed
smoothly, our next choice was the use of an allyl protecting group. This protecting group can be
removed after oligonucleotide synthesis by treatment with tetrakis(triphenylphosphine)palladium (0),
triphenylphosphine and morpholine [51]. Reaction of allyl alcohol with succinic anhydride (7) in the
presence of DMAP afforded the monoprotected dicarboxylic acid 8 [52]. 2’-Amino-2’-deoxyuridine
(9) was synthesized from 3 according to literature procedures [27, 28]. Coupling of acid 8 with 2’-
amino-2’-deoxyuridine (9) in the presence of DCC and HOBt gave the modified nucleoside 10 [50].
Dimethoxytritylation could be accomplished under standard conditions and the corresponding
derivative 11 was isolated and purified without problems.
Scheme 2: Synthesis of the carboxylate-modified nucleoside building block 11.
O
O
a
O
O
HO
O
O
7
8
O
N
O
N
O
N
HN
HN
HN
O
O
O
O
HO
HO
DMTrO
b
O
c
O
O
O
OH NH
OH NH2
OH NH
O
O
O
O
9
10
11
a) allyl alcohol, DMAP, toluene, reflux, 5h (quantitative); b) 8, DCC, HOBt, DMF, rt, overnight (75%);
c) DMTr-Cl, pyridine, rt, 6 h (78%).
Although oligonucleotides modified with a hydroxyl function on the base, sugar or phosphate
backbone have already been synthesized, no previous method for the introduction of an alcohol
functionality via a C 2’-amide bond has been reported [53-59]. Out of the wide variety of available
protecting groups for the protection of this hydroxyl function [53-57], we chose the acetate group.
Deprotection is then possible by the standard ammonia treatment which is used to remove the
cyanoethyl and base protecting groups with concurrent cleavage of the oligonucleotide from the solid
phase. Reaction of acetic acid with β-propiolactone (12) afforded protected carboxylic acid 13 [60]. As
shown in Scheme 3, carboxylic acid 13 was subsequently coupled with 2’-amino-2’-deoxyuridine (9)
in the presence of DCC and HOBt [50]. Dimethoxytritylation of the 5’-OH group was accomplished
under standard conditions and resulted in amide 15 in satisfactory yield.

Molecules 2007, 12
Scheme 3: Synthesis of the hydroxyl-modified nucleoside building block 15.
120
O
O
a
O
HO
O
O
12
13
O
N
O
N
O
N
HN
HN
HN
O
O
O
O
HO
HO
DMTrO
b
c
O
O
OH NH2
OH NH
O
OH NH
O
O
O
O
O
9
14
15
a) H2SO4, HOAc, rt, 4h (quantitative); b) 13, DCC, HOBt, DMF, rt, overnight (48%); c) DMTr-Cl, pyridine,
rt, overnight (78%).
Conclusions
In summary, three different nucleoside analogues containing protected imidazole, carboxylate and
hydroxyl functionalities on the 2’ position of the sugar moiety − compounds 5, 11 and 15 −
respectively, have been synthesized. The incorporation of the different nucleoside analogues into
oligonucleotides as a new approach towards nucleic acid based serine protease models is currently
under investigation.
Experimental Section
General
All reagents were obtained either from Aldrich, ACROS Organics or Fluka and were used without
prior purification. Dimethylformamide (DMF) and pyridine were purchased from ACROS (dry over
molecular sieves). Toluene was distilled from sodium and MeOH was dried on Mg/I2. Reactions were
performed under argon atmospheres. Analytical TLC was carried out on glass plates precoated with
silica gel (Merck, 60F-254, 0.25 mm) and compounds were visualized using phosphomolybdic acid
(PMA) and KMnO4. Flash chromatography was performed on silica gel (KieselgelMerck, 230-400
mesh, Type 9385, 60 Angström). IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR
spectrometer and bands are quoted in cm-1. 1H-NMR and APT spectra were recorded on a Bruker 500
or 300 MHz spectrometer. The deuterated solvents CDCl3, MeOD, DMSO-d6 were obtained from
Aldrich. Chemical shifts (δ units) are expressed in parts per million (ppm) relative to TMS and the
internal solvent peak was used for calibration. When peak multiplicities are reported, the following
abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet. Coupling constants (J values) are
expressed in Hertz (Hz). The Attached Proton Test (APT) technique was used to assign 13C peaks (C,
CH, CH2, CH3). Mass spectra (ES-MS) were recorded on a quadrupole ion trap LC mass spectrometer

Molecules 2007, 12
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(Thermo Finnigan MAT LCQ mass spectrometer) equipped with electrospray ionization and EI-MS
spectra were acquired on a Hewlett-Packard 5998 A (MS, EI) spectrometer.
2’-Amino-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (4)
2’-Azido-2’-deoxyuridine (3) was coevaporated three times with pyridine and dried overnight
under vacuum. To a solution of 3 (200 mg, 0.74 mmol) in pyridine (6 mL) was added 4,4’-dimethoxy-
tritylchloride (378 mg, 1.11 mmol) and the reaction mixture was stirred at rt for 3 days. MeOH (100
µL) was added at 0 °C and the reaction mixture was stirred for 10 min. The reaction mixture was
evaporated under reduced pressure and EtOAc (10 mL) was added. The organic layer was washed
successively with water (10 mL), saturated NaHCO3 (10 mL) and again with water (10 mL). The
combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel (DCM/MeOH/Et3N, 98:1:1) to
afford 2’-azido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (400 mg, 95%): Rf (DCM + 5% MeOH):
0.34; 1H-NMR (500 MHz, CDCl3) δ 7.97 (1 H, d, J= 8.2 Hz), 7.43-7.33 (9 H, m), 6.91 (4 H, d, J= 8.9
Hz), 6.04 (1 H, d, J= 3.0 Hz), 5.42 (1 H, d, J= 8.2 Hz), 4.54 (1 H, dd, J= 6.12 Hz), 4.23 (1 H, dd, J=
3.0, 5.5 Hz), 4.08 (1 H, m), 3.86 (6 H, s), 3.66 (1 H, AB dd, J= 2.41, 11.3 Hz), 3.55 (1 H, AB dd, J=
2.41, 11.3 Hz), 2.80 (1 H, br s), 1.73 (1 H, br s) ppm; APT (CDCl3, 125 MHz) δ 55.2 (CH3), 61.2
(CH2), 67.1 (CH), 69.6 (CH), 83.1 (CH), 87.2 (C), 87.6 (CH), 102.3 (CH), 113.2 (CH), 127.2 (CH),
127.9 (CH), 128.0 (CH), 129.9 (CH), 130.0 (CH), 134.8 (C), 135.0 (C), 139.3 (CH), 144.0 (C), 149.7
(C), 158.6 (C), 162.2 (C) ppm; IR (KBr plate) ν 3500, 2114, 1689 cm-1; ESI-MS (positive mode) 303.3
+
+
(DMTr) , 573.2 (M + Na) . 2’-Azido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (400 mg, 0.7
mmol) was dissolved in MeOH (7 mL) and Pd/C (32 mg) was added. The flask was equipped with a
H2 balloon and the reaction mixture was stirred at room temperature for 4h. The reaction mixture was
filtered through Celite® to remove the Pd/C and the solvent was removed under reduced pressure. The
residue was purified by column chromatography on silica gel (DCM/MeOH/Et3N, 95:4:1) to afford 4
(250 mg, 66%): Rf (DCM/ MeOH, 95:5) 0.2; 1H-NMR (500 MHz, MeOD) δ 7.81 (1 H, d, J= 8.1 Hz),
7.40 (2 H, d, J= 7.3 Hz), 7.31- 7.28 (6 H, m), 7.23 (1 H, m), 6.87 (4 H, d, J= 8.4 Hz), 5.85 (1 H, d, J=
6.8 Hz), 5.32 (1 H, d, J= 8.1 Hz), 4.28 (1 H, dd, J= 2.93, 5.43 Hz), 4.11 (1 H, m), 3.77 (6 H, s), 3.51 (1
H, dd, J= 5.6, 6.7 Hz); 3.42 (1 H, AB dd, J= 3.3, 10.7 Hz), 3.38 (1 H, AB dd, J= 3.1, 10.1 Hz) ppm;
APT (MeOD, 125 MHz) δ 55.8 (CH3), 59.9 (CH), 64.7 (CH2), 72.8 (CH), 79.0 (C), 79.3 (C), 86.4
(CH), 90.4 (CH), 102.9 (CH), 114.2 (CH), 128.1 (CH), 128.9 (CH), 129.4 (CH), 131.4 (CH), 136.6
(C), 142.2 (CH), 145.9 (C), 160.3 (C) ppm; IR (KBr plate) ν 3375, 2970; 2834, 2352; 1865, 1842,
1694, 1651, 1607, 1510, 1462, 1381, 1251, 1176, 1086, 1034, 829, 791, 702 cm-1; ESI-MS (positive
mode) 303.1 (DMTr) +, 545.3 (M) +, 567.7 (M + Na) +.
3-(1-tertbutoxycarbonyl-imidazol-4-yl)-acrylic acid (2)
A solution of urocanic acid (1, 1 g, 7.2 mmol) in DMF (25 mL) was stirred for 1h and then di-tert-
butyldicarbonate (1.58 g, 7.2 mmol) was added. The reaction mixture was stirred overnight at rt and
1
was then coevaporated with toluene to afford 2 in quantitative yield: H-NMR (300 MHz, MeOD) δ
8.27 (1 H, s), 7.94 (1 H, s), 7.46 (1 H, d, J = 15.6 Hz), 6.42 (1 H, d, J = 15.6 Hz), 1.57 (9 H, s) ppm;

Molecules 2007, 12
122
APT (MeOD, 75 MHz) δ 27.4 (CH3), 85.9 (C), 118.5 (CH), 119.5 (CH), 119.6 (CH), 135.2 (CH),
138.4 (C), 146.3 (C), 167.5 (C) ppm; IR (KBr plate) ν 3144, 2979, 2934, 2802, 2668, 2802, 2668,
2544, 1902, 1758, 1703, 1657, 1563, 1492, 1457, 1384, 1336, 1320, 1302, 1255, 1204, 1148, 989, 841,
656 cm-1; ESI-MS (positive mode) 139.0 (M- Boc)+, 182.8 (M- tBu)+, 238.2 (M) +.
2’-[3-(1-tertbutoxycarbonyl-imidazol-4-yl)acryloyl]-amido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxy-
uridine (5)
A solution of 2 (154 mg, 0.65 mmol) in DMF (2 mL) was cooled to 0 °C. 1-(3-Dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (125 mg, 0.65 mmol) and N-hydroxysuccinimide (75 mg,
0.65 mmol) were added. The mixture was allowed to warm to rt and was stirred overnight. The
reaction mixture was then cooled to 0 °C and 4 (300 mg, 0.54 mmol) was added. The reaction was
allowed to warm to room temperature and stirred for 7h, then H2O was added and the aqueous layer
was extracted with EtOAc (3 x). The combined organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography on silica
1
gel (DCM/MeOH, 97:3) to afford 5 (240 mg, 68%): Rf (DCM/MeOH, 9/1): 0.5; H-NMR (300 MHz,
MeOD) δ 8.25 (1 H, s), 7.93 (1 H, d, J = 8.3 Hz), 7.74 (1 H, s), 7.52-7.23 (10 H, m), 6.91 (4 H, d, J =
8.9 Hz), 6.79 (1 H, d, J = 15.4 Hz), 6.17 (1 H, d, J= 8.5 Hz), 5.34 (1 H, d, J = 8.1 Hz), 5.04 (1 H, dd, J
= 5.4, 8.3 Hz), 4.43 (1 H, m), 4.18 (1 H, m), 3.79 (6 H, s), 3.49 (1H, AB dd, J = 2.7, 10.8 Hz), 3.40
(1H, AB dd, J = 2.7, 10.6 Hz), 1.65 (9 H, s) ppm; APT (MeOD, 75 MHz) δ 28.0 (CH3), 49.9 (C) 55.8
(CH3), 57.3 (CH), 65.1 (CH2), 72.6 (CH), 87.4 (CH), 87.8 (CH), 87.8 (C), 88.5 (C), 103.0 (CH), 114.4
(CH), 120.1 (CH), 121.8 (CH), 128.2 (CH), 129.1, 129.5 (CH), 131.4 (CH), 132.7 (CH), 136.6 (C),
136.9 (C), 140.1 (C), 142.5 (CH), 145.8 (C), 147.8 (C), 152.6 (C), 160.3 (C), 165.9 (C), 168.9 (C)
ppm; IR (KBr plate) ν 3306, 2929, 1766, 1722, 1644, 1614, 1462, 1384, 1254, 1091, 988, 836 cm-1;
ESI-MS (positive mode) 303.3 (DMTr) +, 665.6 (M-Boc) +, 766.0 (M) +.
2’-[3-(imidazol-4-yl]acryloyl]-amido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (6)
A solution of urocanic acid (1, 152 mg, 1.1 mmol) in DMF (5 mL) was cooled to 0 °C. 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (210 mg, 1.1 mmol) and N-hydroxy-
succinimide (127 mg, 1.1 mmol) were added. The mixture was allowed to warm to rt and was stirred
for 5h, then 4 (600 mg, 1.1 mmol) was added and the reaction was stirred overnight at rt. H2O was
added and the aqueous layer was extracted with EtOAc (3 x) and CHCl3 (1 x). The combined organic
layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (DCM/MeOH, 9:1) to afford 6 (338 mg, 45%): Rf
(DCM/ MeOH 9/ 1): 0.2; 1H-NMR (300 MHz, MeOD) δ 7.93 (1 H, d, J = 8.1 Hz), 7.77 (1 H, s), 7.51-
7.23 (11 H, m), 6.91 (4 H, d, J = 8.8 Hz), 6.62 (1 H, d, J = 15.6 Hz), 6.17 (1 H, d, J= 8.3 Hz), 5.34 (1
H, d, J = 8.1 Hz), 5.03 (1 H, dd, J = 5.5, 8.3 Hz), 4.42 (1 H, m), 4.18 (1 H, m), 3.79 (6 H, s), 3.49 (1 H,
AB dd, J = 2.7, 10.8 Hz), 3.40 (1 H, AB dd, J = 2.7, 10.7 Hz) ppm; APT (MeOD, 75 MHz) δ 169.5
(C), 165.9 5 (C), 160.4 (C), 152.6 (C), 145.8 (C), 142.5 (CH), 136.9 (C), 136.6 (C), 131.5 (CH), 131.4
(CH), 129.5 (CH), 129.0 (CH), 128.1 (CH), 118.7 (CH), 114.3 (CH), 103.0 (CH), 88.5 (C), 87.9 (CH),
87.4 (CH), 72.6 (CH), 65.1 (CH2), 57.2 (CH), 55.8 (CH3) ppm; IR (KBr plate) ν 3252, 2925, 1760,

Molecules 2007, 12
123
1652, 1614, 1514, 1463, 1384, 1254, 1178, 1014, 836 cm-1; ESI-MS (positive mode): 303.0 (DMTr) +,
665.8 (M) +.
4-allyloxy-4-oxo-butanoic acid (8)
To a solution of succinic anhydride (7, 2 g 2.0 mmol) in toluene (45 mL) was added 4-dimethyl-
aminopyridine (a catalytic amount). Allyl alcohol (1.36 mL, 2.0 mmol) was added dropwise and the
reaction mixture was refluxed for 5h. The solvent was removed under reduced pressure and the residue
1
was purified by bulb-to-bulb distillation (0.04 mmHg, 140 °C) to afford 8 (quantitative): H-NMR
(300 MHz, CDCl3) δ 5.89 (1 H, dddd, J = 5.6, 5.6, 11.1, 16.8 Hz), 5.30 (1 H, dd, J = 1.4, 17.2 Hz),
5.22 (1 H, dd, J = 1.0, 10.4 Hz), 4.58 (2 H, d, J = 5.7 Hz), 2.65 (4 H, t, J = 3.4 Hz) ppm; APT (75
MHz, DMSO) δ 173.3 (C), 171.7 (C), 132.6 (CH), 117.5 (CH2), 64.4 (CH2), 28.6 (CH2), 28.5 (CH2)
ppm; IR (KBr film) ν 3090, 2940, 1737, 1650, 1415, 1346, 1170, 992, 936, 842, 558 cm-1; ESI-MS
(negative mode) 158.6 (M) +.
2’-[(4-allyloxy-4-oxo)-butanoyl]-amido-2’-deoxyuridine (10)
A solution of 8 (182 mg, 1.15 mmol) in DMF (3 mL) was cooled to 0 °C. N,N’-Dicyclohexyl-
carbodiimide (285 mg, 1.38 mmol) and 1-hydroxybenzotriazole (187 mg, 1.38 mmol) were added. The
mixture was stirred at 0 °C for 30 min and 9 (280 mg, 1.15 mmol) was added. The mixture was
allowed to warm up to rt and stirred overnight. The reaction mixture was filtered to remove the
precipitated DCU and concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (gradient elution DCM/MeOH, 95:5 to 9:1) to afford 10 (347 mg, 75%):
Rf (DCM/MeOH, 8:2): 0.46; 1H-NMR (500 MHz, MeOD) δ 8.00 (1 H, d, J = 8.1 Hz), 6.06 (1 H, d, J =
8.6 Hz), 5.92 (1 H, m), 5.74 (1 H, d, J = 8.1 Hz), 5.30 (1 H, dd, J = 1.5, 17.1 Hz), 5.21 (1 H, dd, J =
1.3, 10.5 Hz), 4.61 (2 H, dd, J = 5.6, 8.5 Hz), 4.56 (1 H, dt, J = 1.3, 5.5 Hz), 4.26 (1 H, dd, J = 1.2, 5.6
Hz), 4.09 (1 H, m), 3.77 (1 H, AB dd, J = 3.1, 12.1 Hz), 3.73 (1 H, AB dd, J = 3.1, 12.1 Hz), 2.59 (4 H,
m) ppm; APT (75 MHz, MeOD) 175.0 (C), 174.1 (C), 166.1 (C), 152.8 (C), 142.7 (CH), 133.7 (CH),
118.3 (CH2), 103.3 (CH), 88.6 (CH), 88.1 (CH), 72.2 (CH), 66.4 (CH2), 62.1 (CH2), 56.9 (CH), 34.8
(CH2), 26.8 (CH2) ppm; IR (KBr plate) ν 3322, 2928, 2850, 2738, 2676, 2481, 2355, 1682, 1626,
1575, 1435, 1398, 1244, 1088 cm-1; ESI-MS (positive mode) 406.0 (M+ Na) +.
2’-[(4-allyloxy-4-oxo)-butanoyl]-amido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (11)
Compound 10 was coevaporated with pyridine (3x) and dried under vacuum overnight. To a
solution of 10 (300 mg, 0.78 mmol) in pyridine (1.5 mL) was added 4,4’-dimethoxytritylchloride (397
mg, 1.17 mmol) and the reaction was stirred at rt for 6h. MeOH (100 µL) was added to the cooled
reaction at 0 °C and stirred for 10 min. The reaction mixture was evaporated under reduced pressure
and EtOAc (10 mL) was added. The organic layer was washed with water (10 mL), saturated NaHCO3
(10 mL) and brine (10 mL). The combined organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by column chromatography on silica
gel (gradient elution: DCM+ 1% Et3N → DCM + 1% Et3N+ 1% MeOH → DCM + 1% Et3N + 3%

Molecules 2007, 12
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1
MeOH) to afford 11 (250 mg, 78%): Rf (DCM/MeOH, 95:5): 0.31, H-NMR (500 MHz, MeOD) δ
7.80 (1 H, d, J = 8.1 Hz), 7.41 (2 H, d, J = 7.4 Hz), 7.31-7.20 (7 H, m), 6.87 (4 H, d, J = 8.3 Hz), 6.05
(1 H, d, J = 8.4 Hz), 5.89 (1 H, m), 5.28 (1 H, d, J = 8.2 Hz), 5.28 (1 H, dd, J = 1.6, 17.0 Hz), 5.16 (1
H, dd, J = 1.3, 10.5 Hz), 4.55 (1 H, m), 4.31 (1 H, d, J = 5.5 Hz), 4.11 (1 H, m), 3.76 (6 H, s), 3.47-
3.43 (2 H, m), 3.42 (1 H, AB dd, J = 2.9, 10.7 Hz), 3.35 (1H, AB dd, J = 2.6, 10.6 Hz), 2.65-2.54 (4 H,
m) ppm, APT (75 MHz, MeOD) 175.0 (C), 174.0 (C), 166.0 (C), 160.4 (C), 152.6 (C), 145.8 (C),
142.4 (CH), 136.9 (C), 136.6 (C), 133.7 (CH), 131.4 (CH), 129.5 (CH), 129.0 (CH), 128.1 (CH), 118.2
(CH2), 114.3 (CH), 88.5 (C), 87.6 (CH), 87.3 (CH), 72.4 (CH), 66.3 (CH2), 65.1 (CH2), 57.1 (CH),
55.8 (CH3), 31.3 (CH2), 30.3 (CH2) ppm; IR (KBr plate) ν 3254, 3055, 2935, 1694, 1607, 1510, 1463,
1383, 1251, 1177, 1091, 1032, 990, 830, 703 cm-1; ESI-MS (positive mode) 303.1 (DMTr) +, 707.9 (M
+ Na) +.
3-Acetoxy-propanoic acid (13)
Sulfuric acid (5 µL) was added to a solution of β-propiolactone (12, 3 mL, 3.44 g, 47.7 mmol) in
glacial acetic acid (14 mL). The mixture was stirred at rt for 4h and the solvent was removed under
reduced pressure. The residue was purified by a bulb-to-bulb distillation (0.4 mmHg, 200 °C) to afford
1
13 (quantitative). H-NMR (300 MHz, CDCl3) δ 4.31 (2 H, t, J = 4.31 Hz), 2.67 (2 H, t, J = 6.2 Hz),
2.02 (3 H, s) ppm; APT (CDCl3, 75 MHz) δ 176.6 (C), 170.6 (C), 59.8 (CH2), 33.7 (CH2), 20.8 (CH3)
ppm; IR (KBr film) ν 3106, 2672, 1744, 1636, 1618, 1429, 1387, 1368, 1240, 1187, 1043, 988, 820,
607 cm-1; MS (EI) m/z (%) 43 (100), 55 (22), 73 (24), 89 (17), 115 (19), 133 (M+, 6).
2’-(3-acetoxypropanoyl)-amido-2’-deoxyuridine (14)
A solution of 13 (170 mg, 1.3 mmol) in DMF (5 mL) was cooled to 0 °C. N,N’-Dicyclohexyl-
carbodiimide (265 mg, 1.3 mmol) and 1-hydroxybenzotriazole (166.5 mg, 1.25 mmol) were added.
The mixture was stirred at 0 °C for 1h and 9 (250 mg, 1.05 mmol) was added. The mixture was stirred
overnight at rt, filtered to remove the precipitated DCU and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (DCM/MeOH, 9:1) to afford 14 (175 mg,
1
48%): Rf (DCM/MeOH, 9:1): 0.21; H-NMR (300 MHz, MeOD) δ 7.92 (1 H, d, J = 8.1 Hz), 5.96 (1
H, d, J = 8.4 Hz), 5.64 (1 H, d, J = 8.1 Hz), 4.59 (1 H, dd, J = 5.6, 8.4 Hz), 4.16 (3 H, m), 3.97 (1 H,
m), 3.69 (1 H, AB dd, J = 2.8, 12.0 Hz), 3.64 (1 H, AB dd, J = 2.8, 12.0 Hz), 2.47 (2 H, m), 1.90 (3 H,
s) ppm; APT (75 MHz, MeOD) 173.5 (C), 172.9 (C), 166.1 (C), 152.7 (C), 142.6 (CH), 103.3 (CH),
88.7 (CH), 88.1 (CH), 72.2 (CH), 63.2 (CH2), 61.4 (CH2), 57.0 (CH), 36.0 (CH2), 20.8 (CH3) ppm; IR
(KBr plate) ν 3444, 1667, 1634, 1557, 1463, 1384 cm-1; MS (EI) m/z (%) 43 (100), 52 (47), 77 (30),
107 (23), 157 (4), 314 (M+- Ac, 1), 359 (M+,1).
2’-(3-acetoxypropanoyl)-amido-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyuridine (15)
Compound 14 was coevaporated three times with pyridine and dried under vacuum for 6h. To a
solution of 14 (175 mg, 0.378 mmol) in pyridine (2 mL) was added 4,4’-dimethoxytritylchloride (192
mg, 0.567 mmol) and the reaction was stirred overnight at rt. MeOH (100 µL) was added to the cooled

Molecules 2007, 12
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reaction mixture at 0 °C and stirred for 10 min. EtOAc (10 mL) was added and the organic layer was
washed successively with water (10 mL), saturated NaHCO3 (10 mL) and brine (10 mL). The
combined aqueous layers were extracted with EtOAc. The combined organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (DCM/MeOH, 95:5) to afford 15 (250 mg, 78%): Rf (DCM/MeOH, 9:1):
1
0.57; H-NMR (500 MHz, MeOD) δ 7.83 (1H, d, J = 8.0 Hz), 7.42 (2 H, d, J = 7.6 Hz), 7.31-7.29 (6
H, m), 7.22 (1 H, m), 6.87 (4 H, d, J = 8.6 Hz), 6.06 (1 H, d, J = 8.3 Hz), 5.28 (1 H, d, J = 8.1), 4.86 (1
H, dd, J= 5.5, 8.3 Hz), 4.32 (1 H, m), 4.28 (2 H, m), 4.11 (1 H, m), 3.76 (6 H, s), 3.43 (1 H, AB dd, J =
2.8, 10.6 Hz), 3.36 (1 H, AB dd, J = 2.9, 10.6 Hz), 2.63-2.53 (2 H, m), 1.99 (3 H, s) ppm; APT (75
MHz, MeOD) 173.6 (C), 172.7 (C), 165.9 (C), 160.3 (C), 152.6 (C), 145.8 (C), 142.4 (CH), 136.8 (C),
131.4 (CH), 129.5 (CH), 129.0 (CH), 128.1 (CH), 114.3 (CH), 102.9 (CH), 88.5 (C), 87.6 (CH), 87.4
(CH), 72.4 (CH), 65.1 (CH2), 61.7 (CH2), 57.1 (CH), 55.8 (CH3), 36.1 (CH2), 20.8 (CH3) ppm, IR
(KBr plate) ν 3328, 3064, 2931, 2836, 1694, 1607, 1506, 1463, 1385, 1252, 1177, 1092, 1034, 986,
829, 757, 703, 583, 555 cm-1; ESI-MS (positive mode) 303.3 (DMTr) +, 676.8 (M + NH4) +.
Acknowledgements
Financial support from Ghent University (BOF 01115203) and FWO Vlaanderen (KAN
1.5.186.03) is gratefully acknowledged. M.C. would like to thank Ghent University for an assistant
position.
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Sample Availability: No samples available
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