2′-: O -Methyl- and 2′- O -propargyl-5-methylisocytidine: Synthesis, properties and impact on the isoCd-dG and the isoCd-isoGd base pairing in nucleic acids with parallel and antiparallel strand orientation

Article abstract of DOI:10.1039/c6ob00622a

Oligonucleotides containing 2′-O-methylated 5-methylisocytidine (3) and 2′-O-propargyl-5-methylisocytidine (4) as well as the non-functionalized 5-methyl-2′-deoxyisocytidine (1b) were synthesized. MALDI-TOF mass spectra of oligonucleotides containing 1b are susceptible to a stepwise depyrimidination. In contrast, oligonucleotides incorporating 2′-O-alkylated nucleosides 3 and 4 are stable. This is supported by acid catalyzed hydrolysis experiments performed on nucleosides in solution. 2′-O-Alkylated nucleoside 3 was synthesized from 2′-O-5-dimethyluridine via tosylation, anhydro nucleoside formation and ring opening. The corresponding 4 was obtained by direct regioselective alkylation of 5-methylisocytidine (1d) with propargyl bromide under phase-transfer conditions. Both compounds were converted to phosphoramidites and employed in solid-phase oligonucleotide synthesis. Hybridization experiments resulted in duplexes with antiparallel or parallel chains. In parallel duplexes, methylation or propargylation of the 2′-hydroxyl group of isocytidine leads to destabilization while in antiparallel DNA this effect is less pronounced. 2′-O-Propargylated 4 was used to cross-link nucleosides and oligonucleotides to homodimers by a stepwise click ligation with a bifunctional azide.

Full text of DOI:10.1039/c6ob00622a

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Page 1 of 42
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2’-O-Methyl- and 2’-O-propargyl-5-methylisocytidine: synthesis, properties and impact
on the isoCd – dG and the isoCd – isoGd base pairing in nucleic acids with parallel and
antiparallel strand orientation†
Sunit K. Janaa, Peter Leonarda, Sachin A. Ingalea and Frank Seela*a,b
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, 48149 Münster, Germany and bLaboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
Barbarastrasse 7, 49069 Osnabrück, Germany
Corresponding author: Prof. Dr. Frank Seela
Phone: +49 (0)251 53 406 500; Fax: +49 (0)251 53 406 857
E-mail: Frank.Seela@uni-osnabrueck.de
Homepage: www.seela.net
† Electronic supplementary information (ESI) available: 1H-13C coupling constants, HPLC
profiles, copies of 1H, 13C, 31P, DEPT-135, and 1H-13C gated-decoupled NMR spectra.
1

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Table of contents entry
The impact of 2’-O-alkyl residues on the stability of iCd – dG and iCd – iGd base pairs was
studied in DNA with parallel and antiparallel chain orientation.
2

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Abstract
Oligonucleotides containing 2’-O-methylated 5-methylisocytidine (3) and 2’-O-propargyl-5-
methylisocytidine (4) as well as the non-functionalized 5-methyl-2’-deoxyisocytidine (1b)
were synthesized. MALDI-TOF mass spectra of oligonucleotides containing 1b are
susceptible to a stepwise depyrimidation. On the contrary, oligonucleotides incorporating 2’-
O-alkylated nucleosides 3 and 4 are stable. This is supported by acid catalyzed hydrolysis
experiments performed on nucleosides in solution. 2’-O-Alkylated nucleoside 3 was
synthesized from 2’-O-5-dimethyluridine via tosylation, anhydro nucleoside formation and
ring opening. The corresponding 4 was obtained by direct regioselective alkylation of 5-
methylisocytidine (1d) with propargyl bromide under phase-transfer conditions. Both
compounds were converted to phosphoramidites and employed in solid-phase oligonucleotide
synthesis. Hybridization experiments resulted in duplexes with antiparallel or parallel chains.
In parallel duplexes, methylation or propargylation of the 2’-hydroxyl group of isocytidine
leads to destabilization while in antiparallel DNA this effect is less pronounced. 2’-O-
Propargylated 4 was used to cross-link nucleosides and oligonucleotides to homodimers by a
stepwise click ligation with a bifunctional azide.
Keywords: Oligonucleotides, base pairing, sugar modified nucleosides, isocytidine, parallel-
stranded DNA
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Introduction
Parallel-stranded (ps) DNA stands for an unique DNA structure embodied by a sugar-
phosphate backbone pointing to the same direction. The ps DNA duplex structure was first
suggested by Pattabiraman1 and realized later by Jovin,2 Seela,3 and others.4,5 DNA with
parallel strand orientation can be generated from single-stranded DNA consisting of the four
nucleosides 2’-deoxyisocytidine (iCd, 1a) or its 5-methyl-derivative (MeiCd, 1b), 2’-
deoxyisoguanosine (iGd, 2a) as well as dA and dT (Fig. 1). As the recognition site of 2’-
deoxyisocytidine (1a) is similar to that of 2’-deoxyguanosine, nucleoside 1a base pairs with
2’-deoxyguanosine in DNA with parallel strand orientation (ps DNA) and with 2’-
deoxyisoguanosine in DNA duplexes with antiparallel strands (aps DNA) (for base pair
motifs see Fig. 2).3-5 Enzymatic recognition, spectroscopic as well as drug-binding properties
of ps DNA differ from that of native DNA with antiparallel chains.3h According to spin-label
experiments, nucleobase distances within one helix turn are 5-10% longer in ps DNA than in
aps DNA.3i DNA with parallel strand orientation is less stable than its antiparallel congener,3-
5 due to weaker stacking interactions and the weaker hydrogen bonding of the dA·dT reverse
Watson-Crick (Donohue) base pair. However, the tridentate iGd-dC and iCd-dG base pairs are
used to stabilize ps DNA. In aps DNA, the iGd-iCd base pair is even more stable than the
canonical dG-dC pair.
Fig. 1 Structures of isocytosine and isoguanine nucleosides.
4

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Fig. 2 Base pairs3k of dA-dT, iGd-dC, dG-MeiCd and iGd-MeiCd in DNA duplexes and DNA-
RNA hybrids with parallel (ps) and antiparallel (aps) strand orientation. Arrows indicate
strand orientation.
Until today, only a few reports are describing the functionalization at the 2’-position of
isocytidine (1a-f, Fig. 1) or isoguanosine (2a-d) in the isoguanosine-isocytidine base
pair.3a,b,6,7 Only recently, modification at the 2’-position of 2’-deoxyisoguanosine (2a) by
fluoro (2c) and propargyloxy (2d) substituents has been reported.3a,b However, very little is
known on 2’-functionalization of 2’-deoxyisocytidine (1a) and its 5-methyl derivative 1b.3a,6
2’-Deoxyisocytidine (iCd, 1a) is a structural analog of the canonical DNA constituent 2’-
deoxycytidine. Different to 2’-deoxycytidine, 2’-deoxyisocytidine (1a) is rather labile under
acidic conditions.3e To this end, 5-methyl-2’-deoxyisocytidine (1b) is used as 2’-
deoxyisocytidine (1a) substitute.5c However, also the 5-methyl derivative 1b decomposes in
acidic environment more easily than the canonical pyrimidine nucleosides.3c
In this manuscript, the synthesis of 2’-O-5-dimethylisocytidine (3) and 5-methyl-2’-O-
propargylisocytidine (4) and their building blocks for oligonucleotide solid-phase synthesis is
described. Both are expected to be more stable under acidic conditions than the 2’-
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deoxyribonuclesides 1a,b. As the selective alkylation of the 2’-position of canonical
nucleosides is often encountered with difficulties and side products (3’-O-isomers) are
formed during alkylation,12 the synthesis of 2’-O-5-dimethylisocytidine (3) is based on the
conversion of the commercially available 2’-O-5-dimethyluridine.8,9 For the corresponding 5-
methyl-2’-O-propargylisocytidine (4) the direct alkylation of isocytidine is used since a
suitable propargylated precursor is not easy accessible. Oligonucleotides containing 3 and 4
were synthesized. Hybridization properties and thermal stability of DNA/RNA hybrids and
DNA/DNA duplexes with parallel and antiparallel strand orientation were studied.
Results and discussion
Synthesis of nucleosides and building blocks
The synthesis of 2’-O-5-dimethylisocytidine (3) was performed by two different routes.
Synthesis procedure (A) uses 2’-O-5-dimethyluridine (5)9 as starting material (Scheme 1) and
follows a similar route as it was reported for 2’-deoxyisocytidine10a,5d and 2’-modified
isocytidine nucleosides like 2’-O-methylisocytidine (1e)6 and 2’-deoxy-2’-fluoroisocytidine
(1f)3a. Initially, nucleoside 5 was tosylated with p-toluene sulfonyl chloride in pyridine.
Different to the reaction of 2’-deoxyuridine which yields the 5’-O-tosylated compound as
major product (60%),10b but in accordance to the tosylation of 2’-deoxy-2’-fluorouridine,3a a
mixture of three reaction products was formed (5’-O-tosylated compound 8 (34%), 3’-O-
derivative 7 (14%), 3’,5’-bis-O-tosylated nucleoside 6 (21%)). The reaction products were
separated by flash column chromatography and characterized by NMR spectra and ESI-TOF
mass spectra. Next, the 5’-O-tosylated 8 was treated with DBU (1,8-
diazabicyclo[5.4.0]undec-7-en) in dry CH3CN under reflux conditions (1 h, TLC-monitoring)
affording 9 (53%). Finally, treatment of the 2,5’-anhydro nucleoside 9 with saturated
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NH3/MeOH (100 °C for 6 h) furnished 2’-O-methyl-5-methylisocytidine (3) in 52% yield
(Scheme 1; for details, see Experimental section).
Scheme 1 Synthesis of 2’-O-5-dimethylisocytidine (3) via sulfonylation of 2’-O-5-
dimethyluridine (5). Reagents and conditions: (i) p-toluene sulfonyl chloride, pyridine, 16 h,
rt; (ii) DBU, CH3CN, reflux, 1 h; (iii) sat. NH3/MeOH, 6 h, 100 °C.
In route (B) a protection/deprotection protocol was employed (Scheme 2). To this end,
compound 12 was synthesized first from compound 5 according to a literature protocol.11
Then, tosylation of 12 with p-toluene sulfonyl chloride gave 13 in 77% yield. Conversion of
13 to cyclonucleoside 14 with DBU proceeded in moderate yield (50%). Finally, cleavage of
the acetyl group gave 9 (81%), which was converted to 3 as described above. In summary,
the overall yields for both routes are comparable [9.4% (A), 12.8% (B)]. According to the
lower number of reactions steps, route A is favoured.
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Scheme 2 Synthesis of 2’-O-5-dimethylisocytidine (3) via sugar protection and deprotection.
Reagents and conditions: (i) 4,4’-dimethoxytrityl chloride, pyridine, rt; (ii) acetic anhydride,
pyridine, rt; (iii) AcOH/H2O (4:1), rt; (iv) p-toluene sulfonyl chloride, pyridine, rt; (v) DBU,
CH3CN, reflux, 30 min; (vi) K2CO3, MeOH, rt; (vii) sat. NH3/MeOH, 6 h, 100 °C.
Various approaches have been attempted to propargylate ribonucleosides selectively at the
2’-position. Often, inseparable mixtures of 2’/3’-isomers are formed.12 In a few cases the
3’,5’-hydoxyl groups were protected before propargylation.13 We found that isocytidine14
can be propargylated in a regioselective way. To this end, phase-transfer conditions using
propargyl bromide together with NaH and tetrabutylammonium iodide (TBAI) were used.
However, at elevated temperature (55 °C) the yield of nucleoside 4 was unsatisfactory due to
formation of several side products. Nevertheless, when the reaction was performed at 0 °C to
room temperature for 12 h as described by Hall for cytidine,15 2’-O-propargylated 5-
methylisocytidine 4 was isolated in 62% yield (Scheme 3).14 Formation of the 3’-O-
propargylated isomer was not observed. The structure of 4 was confirmed by 1H-1H Cosy,
HSQC and HMBQC spectra (for spectra, see the ESI†).
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Scheme 3 Synthesis of 2’-O-propargyl-5-metylisocytidine 4. Reagents and conditions: (i)
triphenylphosphine, diethylazodicarboxylate, THF, 20 min, rt; (ii) sat. NH3/MeOH, 16 h, 110
°C; (iii) propargyl bromide, NaH, TBAI, DMF, 0°C to rt, 12 h.
Then, nucleosides 3 and 4 were converted to building blocks for solid-phase oligonucleotide
synthesis (Scheme 4). Protection of the 2-amino group of 3 and 4 with a
dimethylaminomethylidene residue gave 17 (85%) and 18 (72%), respectively. 4,4’-
Dimethoxytritylation of the 5’-OH groups afforded 19 (81%) as well as 20 (77%). Finally,
phosphitylation of the DMT compounds yielded the phosphoramidites 21 and 22 in 80% and
87% yield, respectively.
Scheme 4 Synthesis of the phosphoramidites of 2’-O-alkylated-5-methylisocytidines.
Reagents and conditions: (i) N,N-dimethylformamide dimethyl acetal, MeOH, rt; (ii) 4,4’-
dimethoxytrityl chloride, pyridine, rt; (iii) NC(CH2)2OP(Cl)N(i-Pr)2, DIPEA, DCM, rt.
All synthesized compounds were characterized by 1H-, 13C- NMR spectra as well as ESI-TOF
mass spectra (see Experimental section). DEPT-135 and 1H-13C gated-decoupled or 1H-13C
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correlated (HMBC, HSQC) NMR spectra were used to assign the 13C NMR signaDlsOI(:T10a.1b03lVe9i/esCw6AOrtBic0le0O6n2li2nAe
S1† and S2†). For details see the Experimental section (for spectra, see the ESI†).
Synthesis and characterization of oligonucleotides
Oligonucleotides (ODNs) were prepared by solid-phase synthesis using the phosphoramidites
of 1b, 2a as well as 21 and 22 together with the canonical phosphoramidites and applying the
standard protocol for solid-phase oligonucleotide chemistry. Oligonucleotides were cleaved
from CPG and deprotected in concentrated aqueous NH3 at 55 °C for 16 h. The coupling
yields of the modified building blocks were always higher than 95%. All synthesized
oligonucleotides were purified by reversed-phase HPLC (RP-18), detritylated with 2.5%
dichloroacetic acid in dichloromethane and again purified by HPLC. The contents of single
peaks were isolated in all cases (Fig. S2†). Subsequently, the molecular masses were
determined by MALDI-TOF mass spectra. Table 1 displays all modified oligonucleotides
used in this study together with their mass data.
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Table 1 Synthesized oligonucleotides and their molecular masses measured by MDAOLI: 1D0.1I0-3T9O/C6FOB00622A
mass spectra
M.W. calcd.a
M.W. foundb
M.W. calcd.a
M.W. foundb
Oligonucleotides
Oligonucleotides
3645.4
3644.9
3645.4
3645.5
5’-d(TAiGiGTCAATACT) (ODN-3)
5’-d(TAiGGTCAATACT) (ODN-11)
3673.5
3674.4
3659.4
3660.5
5’-d(AGTATTGAMeiCMeiCTA) (ODN-4)
5’-d(AGTATTGACMeiCTA) (ODN-12)
3734.5
3733.4
3689.5
3689.3
5’-d(AGTATTGAMeiCOMeMeiCOMeTA) (ODN-5)
5’-d(AGTATTGAMeiCOPropMeiCOPropTA) (ODN-6)
5’-d(ATMeiCMeiCAiGTTATiGA) (ODN-7)
5’-d(ATMeiCOMeMeiCOMeAiGTTATiGA) (ODN-8)
5’-d(ATMeiCOPropMeiCOPropAiGTTATiGA) (ODN-9)
5’-d(AGTATTGACMeiCOMeTA) (ODN-13)
5’-d(AGTATTGACMeiCOPropTA) (ODN-14)
5’-d(ATMeiCOMeMeiCAiGTTATiGA) (ODN-15)
5’-d(ATMeiCOPropMeiCAiGTTATiGA) (ODN-16)
3781.6
3781.9
3781.6
3781.9
3673.5
3673.3
3703.6
3706.0
3733.5
3733.7
3727.5
3726.9
3781.6
3781.2
3901.6
3903.8
5’-d(AGTATTGACMeiCAzidoTA) (ODN-17)
5’-d(AGTATTGACMeiCTA)
3781.5
3781.3
7614.1
7612.7
5’-(UAGGUCAAUACU) (ODN-10)
│
(ODN-18)
5’-d(AGTATTGACMeiCTA)
a Calculated on the basis of molecular weight as [M+H]+. b Determined by MALDI-TOF mass spectrometry as [M+H]+ in
the linear positive mode.
MALDI-TOF mass spectral identification of oligonucleotides is limited by the stability of
monomers. In particular, the more acid sensitive purine nucleosides can be hydrolysed at the
N-glycosylic bond while pyrimidine nucleosides are usually stable. The use of 7-deazapurine
residues in place of purines overcomes the “purine” problem.16 Only little or no
fragmentation of oligonucleotides is observed by using 3-hydroxypicolinic acid (3-HPA) as
matrix. Surprisingly, MALDI-TOF spectra of oligonucleotides containing 5-methyl-2’-
deoxyisocytidine (1b) show strong fragmentation peaks even when 3-HPA is used as matrix.
This is demonstrated in Fig. 3a showing the spectrum of the oligonucleotide 5’-
d(T1bATAA1bT1b1bTA) incorporating four MeiCd (1b) residues. Besides the peak
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corresponding to the calculated molecular mass of the oligonucleotide (3623 Da),DaOdI:d10it.1i0o3n9/aCl6OB00622A
peaks appear with lower masses (3514 Da, 3407 Da, 3301 Da). These data comply with
oligonucleotides that have lost one or more 5-methylisocytosine residues in a stepwise
manner thereby forming “apyrimidinic acids” (Fig. 3b).17
According to the mechanism of depurination and depyrimidation18b,c, protonation of the
nucleobase or the ring oxygen of the sugar moiety takes place and an oxocarbenium ion is
formed.18 Then, a water molecule is added (route 1, Fig. S3†) or elimination takes place
(route 2, Fig. S3†). According to the mass data, addition of water (route 1) is the dominant
reaction in case of MeiCd (1b) cleavage.
Fig. 3 a) MALDI-TOF mass spectra of 5’-d(T1bATAA1bT1b1bTA) measured with 3-HPA
as matrix, b) plausible calculation for the mass difference observed in the MALDI-TOF
spectra.
In order to verify the MALDI data of oligonucleotides, the pKa values of 2’-deoxyisocytidine
1b, the ribonucleoside 1c as well as of the 2’-O-methyl derivative 3 were determined
spectrophotometrically at 262 nm. The UV curves show isosbestic points (Fig. 4a). All three
isocytidine nuclesosides have pKa values between 4.2 and 4.4 (Fig. 4b-d). These values are
very similar to that of dC (pKa = 4.5). Consequently, the pKa value cannot account for the
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hydrolytic instability of iC nucleosides. Generally, two mechanisms are possible for the
proton catalysed glycosylic bond cleavage with protonation of the nucleobase or the ring
oxygen of the sugar moiety as initial step. If the nucleobase is protonated first, the 2’-
deoxyisocytidine base has to be a better leaving group than the 2’-deoxycytidine base. In case
of protonation of the ring oxygen of the sugar moiety, an achimeric assistance of the
protonated iC amino group has to be considered.
a)
b)
c)
d)
Fig. 4 a) UV spectral changes of nucleoside 1b at various pH values; b-d) titration profiles of
1b (b), 1c (c) and 3 (d) measured at 262 nm and plotted vs pH-values. First derivatives
indicate pKa values.
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In order to quantify the low acid stability of nucleosides 1b, the glycosylic bond hydrolysis
was performed in solution (0.1 N HCl, room temperature) and monitored by HPLC (Fig. 5a).
By this means, a half-life value of 190 min was determined for 1b (Fig. 5a) and even at pH
5.0 “depyrimidination” was observed (Fig. 5b). For comparison, the most labile canonical
purine nucleoside dA was measured. Its half-life is 80 min (Fig. S2†). So, dA is even less
stable than MeiCd (1b). Consequently, dA residues of oligonucleotides should be easier
depurinated during MALDI-TOF measurement than the nucleoside 1b residues. As this is not
the case, degradation of 1b in MALDI-TOF might occur at the excited state under the action
of light. This phenomenon might be used to identify the number of 1b residues of DNA
oligonucleotides. Most interestingly, this degradation is not observed for oligonucleotides
incorporating 2’-O-methyl nucleoside 3 or the 2’-O-propargyl derivative 4 as well as 2’-
deoxy-2’-fluoroisocytidine (1f) (Fig. S1†). They are stable under MALDI-TOF conditions as
well as in solution and can be identified and handled without any problems. The electron
withdrawing properties of 2’-O-alkoxy substituents reduce the chance of protonation and
provide the sensitive MeiCd moiety more stability against hydrolysis.
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(a)
(b)
(c)
Fig. 5 a) Reversed-phase HPLC elution profile of MeiCd (1b) after treatment (90 min) with
0.1 N HCl at rt, monitored at 260 nm using an isocratic mixture of 0.1 M TEAA/MeCN 99:1;
b) acid-catalyzed hydrolysis of MeiCd (1b) and MeiCOMe (3) followed by RP-18 HPLC. %
corresponds to peak area integration of starting material; c) proton-catalyzed degradation of
1b measured in Na-cacodylate buffer (pH 5.0), 0.1 M NaCl at 60 °C. The reaction was
followed UV-spectrophotometrically (for details, see the Experimental section).
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Base pairing and functionalization of oligonucleotides
Various manuscripts are describing the impact of 2’-O-methyl substituents on the stability of
oligonucleotides with dA-dT or dG-dC base pairs.19 Now, we studied the effect of 2’-
alkylation on the iGd- MeiCd base pair in aps DNA and the dG-MeiCd pair in ps DNA (Fig. 2)
as well as in DNA/RNA hybrids. It is well established that the iGd-MeiCd base pair formed in
aps DNA is more stable than the dG-dC pair (Table 2).5c The same is valid for the iG-iC pair
in ribooligonucleotides.20 The corresponding MeiCd-dG base pair in ps DNA leads to ps
duplexes which are generally less stable than those with aps strand orientation.
Table 2 displays the Tm values and thermodynamic data of aps and ps DNA duplexes as well
as ps DNA-RNA hybrids with single dG-iCd or iGd-iCd base pair incorporation with and
without sugar modification. Table 3 summarizes data for oligonucleotides with two
consecutive modifications. A homodimer cross-linked via the 2’-propargyl residues was also
studied (Table 3). From the results displayed in Tables 2 and 3, the following conclusions can
be drawn:
(i) In aps DNA, 2’-O-alkylation of the ribofuranose moiety of iCd within the iGd-iCd base pair
with a methyl or a propargyl residue leads to a thermal stability which is higher compared to
the dG-dC pair but slightly lower than for the non-functionalized iGd-iCd pair. This is in line
with 2’-O-methylated oligonucleotides with canonical dA-dT or dG-dC base pairs showing
similar helix stability as corresponding 2’-deoxyribooligonucleotides. Nevertheless, stability
changes are sequence dependent and can result in higher or lower Tm values.19
(ii) The situation is different for ps DNA. Methylation or propargylation of the 2’-hydroxyl
group leads to significant destabilization of duplexes. Apparently, 2’-alkoxy groups are not
well accommodated in the parallel DNA double helix. A similar effect was observed for 2'-
fluoro nucleosides and might result from the preferred N-conformation of those nucleosides;
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a structural property which is not well accepted by ps DNA due to its special backbone
structure.
(iii) In ps DNA/RNA hybrids the destabilization of the 2’-O-alkylation is less pronounced
compared to ps DNA/DNA duplexes.
(iv) Consecutive incorporation of two 2’-O-alkylated base pairs in aps as well as in ps DNA
has a negative impact on the stability of DNA duplexes as well as on DNA/RNA hybrids.
This might result from steric crowding of the 2’-alkoxy substituents and/or the more
lipophilic character of the modified sugar residues.
(v) In oligonucleotide duplexes 5-methylisocytosine nucleosides show a strong
discrimination against dG in comparison to iGd. Two mispair incorporations result in a strong
Tm decrease (Table 3).
(vi) Regarding thermodynamic data, the stability changes observed for aps DNA with sugar
modified base pairs is mainly of enthalpic origin while in ps DNA duplexes entropic changes
play a certain role.
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Table 2 Tm Values and thermodynamic data of 2’-O-alkylated 12-mer duplexes and DNA-
RNA hybrids with parallel and antiparallel strand orientation and single modificationsa
Tm
[°C]b
∆Tm
[°C]
∆H°
∆S°
∆G°310
APS DNA DUPLEXES
[kcal/mol] [cal/K-1mol-1] [kcal/mol]
5’-d(TAGGTCAATACT) (ODN-1)
3’-d(ATCCAGTTATGA) (ODN-2)
47
52
49
-
-69
-85
-80
-188
-235
-222
-10.1
-11.7
-10.9
5’-d(TA iGGTCAATACT) (ODN-11)
3’-d(ATMeiCCAGTTATGA) (ODN-12)
+5 c)
+2 c)
5’-d(TA iG GTCAATACT) (ODN-11)
3’-d(ATMeiCOMeCAGTTATGA) (ODN-13)
5’-d(TA iG
G TCAATACT) (ODN-11)
48
50
+1 c)
+3
-76
-78
-210
-216
-10.6
-11.2
3’-d(ATMeiCOPropCAGTTATGA) (ODN-14)
5’-d(TA iG
3’-d(ATMeiCAzidoCAGTTATGA) (ODN-17)
G TCAATACT) (ODN-11)
PS DNA DUPLEXES
5’-d(TA
5’-d(ATMeiCMeiCAiGTTATiGA) (ODN-7)
5’-d(TA GTCAATACT) (ODN-1)
5’-d(ATMeiCOMeMeiCAiGTTATiGA) (ODN-15)
G
GTCAATACT) (ODN-1)
36
27
27
-
-61
-56
-54
-171
-161
-154
-7.7
-6.0
-6.1
G
-9 d)
-9 d)
5’-d(TA
G
G TCAATACT) (ODN-1)
5’-d(ATMeiCOPropMeiCAiGTTATiGA) (ODN-16)
PS DNA/RNA HYBRIDS
5’- (UA
G GUCAAUACU) (ODN-10)
34
31
29
-
-56
-57
-54
-157
-161
-154
-7.4
-6.7
-6.5
5’-d(ATMeiCMeiCAiGTTATiGA) (ODN-7)
5’- (UA G UCAAUACU) (ODN-10)
5’-d(ATMeiCOMeMeiCAiGTTATiGA) (ODN-15)
5’- (UA G UCAAUACU) (ODN-10)
5’-d(ATMeiCOPropMeiCAiGTTATiGA) (ODN-16)
G
-3 e)
-5 e)
G
a Measured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, 10 mM Na-cacodylate (pH 7.0) with 5 µM + 5 µM single strand
concentration. b Tm values were determined from the melting curves by using the software MELTWIN 3.0. c-e ∆Tm was
calculated as Tm modified duplex − Tm unmodified duplex 1·2c , 1·7d or 10·7e. Thermodynamic data were determined from
van’t Hoff plots with concentration-dependent Tm values and were consistent with those obtained from curve fitting within
10%. The van’t Hoff data for the formation of the duplex ODN-1 • ODN-2 in the above mentioned buffer are as follows:
∆H° = - 76 kcal/mol, ∆S° = -212 cal/K mol, ∆G°310 = -10.2 kcal/mol.
18

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Organic & Biomolecular Chemistry
Table 3 Tm values and thermodynamic data of 2’-O-alkylated 12-mer DNA dupleDxOeIs: 10a.n10d3V9i/eCw6AOrtBic0le0O6n2li2nAe
DNA-RNA hybrids with parallel and antiparallel strand orientation and consecutive
modifications a
Tm
[°C]b
∆Tm
[°C]
∆H°
∆S°
∆G°310
[kcal/mol]
APS DNA DUPLEXES
[kcal/mol] [cal/K-1mol-1]
5’-d(TAGGTCAATACT) (ODN-1)
3’-d(ATCCAGTTATGA) (ODN-2)
47
-69
-188
-10.1
-
5’-d(TA iG iGTCAATACT) (ODN-3)
3’-d(ATMeiCMeiCAGTTATGA) (ODN-4)
54
43
42
34
27
30
+7 c)
-4 c)
-93
-75
-74
-56
-50
-49
-261
-210
-208
-181
-141
-135
-12.8
-9.4
-9.0
-7.2
-6.2
-6.8
5’-d(TA
iG
iG
TCAATACT) (ODN-3)
3’-d(ATMeiCOMeMeiCOMeAGTTATGA) (ODN-5)
5’-d(TA
iG
iG
TCAATACT) (ODN-3)
-5 c)
3’-d(ATMeiCOPropMeiCOPropAGTTATGA) (ODN-6)
5’-d(TA G GTCAATACT) (ODN-1)
3’-d(ATMeiCMeiCAGTTATGA) (ODN-4)
-13 c)
-20 c)
-17 c,f)
5’-d(TA
G
G
TCAATACT) (ODN-1)
3’-d(ATMeiCOMeMeiCOMeAGTTATGA) (ODN-5)
5’-d(TA
G
G
TCAATACT) (ODN-1)
3’-d(ATMeiCOPropMeiCOPropAGTTATGA) (ODN-6)
PS DNA DUPLEXES
5’-d(TA
G
GTCAATACT) (ODN-1)
36
-
-61
-171
-135
-124
-7.8
-5.8
-5.9
5’-d(ATMeiCMeiCAiGTTATiGA) (ODN-7)
5’-d(TA TCAATACT) (ODN-1)
G
G
24 f)
23 f)
-12 d)
-13 d)
-48 f)
-44 f)
5’-d(ATMeiCOMeMeiCOMeAiGTTATiGA) (ODN-8)
5’-d(TA
G
G
T CAATACT) (ODN-1)
5’-d(ATMeiCOPropMeiCOPropAiGTTATiGA) (ODN-9)
PS DNA/RNA HYBRIDS
5’- (UA
G GUCAAUACU) (ODN-10)
34
28
28
-
-56
-57
-48
-157
-163
-134
-7.4
-6.3
-6.5
5’-d(ATMeiCMeiCAiGTTATiGA) (ODN-7)
5’-( UA G U CAAUACU) (ODN-10)
G
-6 e)
-6 e)
5’-d(ATMeiCOMeMeiCOMeAiGTTATiGA) (ODN-8)
5’-( UA
G
G
U CAAUACU) (ODN-10)
5’-d(ATMeiCOPropMeiCOPropAiGTTATiGA) (ODN-9)
CROSS-LINKED HOMODIMER
5’-d(TA iG GTCAATACT) (ODN-11)
3’-d(ATMeiCCAGTTATGA)
47
-
-75
-208
-10.4
│
(ODN-18)
3’-d(ATMeiCCAGTTATGA)
5’-d(TA iGG TCAATACT) (ODN-11)
a Measured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, 10 mM Na-cacodylate (pH 7.0) with 5 µM + 5 µM single strand
concentration. b Tm values were determined from the melting curves by using the software MELTWIN 3.0. c-e ∆Tm was
calculated as Tm modified duplex − Tm unmodified duplex 1·2c , 1·7d or 10·7e. Thermodynamic data were determined from
van’t Hoff plots with concentration-dependent Tm values and were consistent with those obtained from curve fitting within
10%. The van’t Hoff data for the formation of the duplex ODN-1 • ODN-2 in the above mentioned buffer are as follows:
∆H° = - 76 kcal/mol, ∆S° = -212 cal/K mol, ∆G°310 = -10.2 kcal/mol. f Data were calculated from incomplete melting
profiles.
19

Organic & Biomolecular Chemistry
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Besides the Tm modulating effect of the 2’-alkoxy groups, the 2’-O-propargyl subDstOiIt:u10e.1n0t3V9i/eCw6AOrtBic0le0O6n2li2nAe
offers the opportunity to cross-link two DNA strands21 by the stepwise click reaction using
the bifunctional azide 25.22 Consequently, the application of the 2’-O-propargyl group as a
clickable site for cross-linking was studied. The cross-link reaction was performed on the
propargylated nucleoside 4 as well as on the oligonucleotide ODN-14 containing nucleoside
4 in each strand. First, the copper(I) catalyzed click reaction was performed on nucleoside 4
in THF/t-BuOH/H2O together with an five-fold excess of the bis-azide 25, copper(II)sulfate
and Na-ascorbate as reducing agent to afford the mono-functionalized nucleoside 23 in 66%
yield.23 Then, the azido-functionalized nucleoside 23 was used in a second click reaction with
copper(II)sulfate and sodium ascorbate. Here, an equal amount of the azido compound 23 and
propargylated iCd 4 was used to obtain the bis-nucleoside 24 in 64% yield (Scheme 5).
Scheme 5 Synthesis of bis-nucleoside 24 using the stepwise click reaction. Reagents and
conditions: (i) CuSO4·5H2O, sodium ascorbate, ratio 4/25 (1/5), THF/t-BuOH/H2O (3:1:1,
v/v/v), 1 h, rt; (ii) CuSO4·5H2O, sodium ascorbate, ratio 4/23 (1/1), THF/t-BuOH/H2O (3:1:1,
v/v/v), 16 h, rt.
Then, the copper(I) catalyzed click reaction was performed on oligonucleotide ODN-14
containing the propargylated iCd derivative 4. For mono-functionalization an excess of the
bis-azide 25 (ODN-14 to azide 25, 1/15) was used leading to the mono-functionalized
oligonucleotide ODN-17 bearing one azido group (Scheme 6). Then, a second click reaction
20

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Organic & Biomolecular Chemistry
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DOI: 10.1039/C6OB00622A
was performed using ODN-17 and the propargylated ODN-14 in an equimolar ratio yielding
the cross-linked homodimer ODN-18.
Scheme 6 Stepwise click reaction of propargylated isocytidine DNA with symmetrical bis-
azide 25.
The purified homodimer was characterized by MALDI-TOF mass spectrometry (Table 1).
Then, the homodimer was hybridized with two complementary strands to form two double
helices cross-linked by the bis-azido linker 25 (Fig. 6b). The thermal stability of the cross-
linked aps duplex is similar (Tm = 47°C) to the corresponding individual duplexes
incorporating 2’-O-propargylated isocytidine (Tm = 48°C, Table 2).
Circular dichroism can be used to distinguish between the structures of right-handed duplexes
such as A- and B-DNA in solution.25 B-DNA has a conservative CD spectrum with a
negative Cotton effect at around 250 nm and a positive loop at 275 nm and a cross-over band
around 260 nm. In order to show that the two helices connected by a bis-click linker have a
similar structure as the non-ligated molecules, the CD spectra of ODN-1∙ODN-2, the duplex
ODN-11∙ODN-12 containing one 5-methyl-2’-deoxyisocytidine-iGd base pair, the 2’-O-
21

Organic & Biomolecular Chemistry
Page 22 of 42
propargylated duplex ODN-11∙ODN-14, the azido-functionalized duplex ODN-11∙ODN-V1ie7w Article Online
DOI: 10.1039/C6OB00622A
and the cross-linked duplex ODN-11∙ODN-18∙ODN-11were measured (Fig. 6a). In all cases,
the shape of the CD spectra with a negative loop (~250 nm) and a positive loop (~275 nm)
corresponds to a B-DNA structure. Thus, it can be concluded that functionalization of the 2’-
O-position of isocytidine with alkyl residues or cross-linking of two identical strands has no
significant influence on the overall structure of the DNA double helices. Consequently, the
propargyl residue of iCd is a versatile linker unit to ligate DNA double helices and generate
structures related to four-way junctions.24
(a)
(b)
Fig. 6 (a) CD spectra of DNA duplexes and cross-linked helices. Each sample contained 0.5
+ 0.5 A260 units in 1 mL 0.1 M NaCl, 10 mM MgCl2, 10 mM Na-cacodylate (pH 7.0) buffer.
(b) Schematic presentation of DNA duplexes and the cross-linked homodimer (red)
hybridized with its complementary oligonucleotides (blue). For the structure of the linker see
Scheme 6.
Conclusion
2’-O-5-Dimethylisocytidine 3 was synthesized from 2’-O-5-dimethyluridine 5 or 3’-O-
acetylated 5 via tosylation, anhydronucleoside formation and ring opening. The
corresponding 2’-O-propargyl-5-methylisocytidine (4) was prepared by direct alkylation of 5-
methylisocytidine with propargylbromide under phase transfer conditions at ambient
22

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Organic & Biomolecular Chemistry
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temperature. The method was regioselective for the 2’-isomer and gave nucleosidDeO4I:.10.1039/C6OB00622A
Oligonucleotides containing non-functionalized 5-methyl-2’-deoxyisocytidine (1b) are not
stable during MALDI-TOF mass spectrometric analysis and are susceptible to
depyrimidation; corresponding oligonucleotides containing 2’-O-alkylated isocytidines 3 and
4 are stable. Moreover, the 2’-alkoxy functionalization at the 2’-ribo “down” position
stabilizes the molecule against glycosylic bond hydrolysis and does not lead to unwanted
cyclonucleoside formation as observed for the corresponding 2’-fluoro nucleoside.
Oligonucleotide duplexes containing MeiCd-iGd or MeiCd-dG base pairs and 2’-functionalized
isocytidine residues gave access to aps and ps DNA. In parallel duplexes, methylation or
propargylation of the 2’-hydroxyl group of isocytidine leads to destabilization while in
antiparallel DNA this effect is less pronounced. The triple bond of the 2’-O-propargylated iCd
(4) was used to produce oligonucleotide cross-links. To this end, the bis-azide 25 was
employed in a stepwise click reaction. Homodimeric oligonucleotides were synthesized and
hybridized with complementary strands to yield double helices linked by the bis-azide 25
maintaining B-DNA structure.
Experimental section
General methods and materials
All chemicals and solvents were of laboratory grade as obtained from commercial suppliers
and were used without further purification. Thin-layer chromatography (TLC) was performed
on TLC aluminum sheets covered with silica gel 60 F254 (0.2 mm). Flash column
chromatography (FC): silica gel 60 (40-60 μM, for flash chromatography) at 0.4 bar. UV
23

Organic & Biomolecular Chemistry
Page 24 of 42
spectra: λmax (ε) in nm, ε in dm3 mol-1 cm-1. NMR spectra were measured at 300.1D5OMI: 10H.10z3V9fio/eCwr6AOrtBic0le0O6n2l2inAe
1H, 75.48 MHz for 13C and 121.52 MHz for 31P. The J values are given in Hz; δ values are
given in ppm relative to Me4Si as internal standard. For NMR spectra recorded in DMSO-d6,
the chemical shift of the solvent peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for
13C NMR. The 13C NMR signals were assigned on the basis of DEPT-135 and 1H-13C gated-
decoupled NMR spectra (Tables S1† and S2†). Reversed-phase HPLC was carried out on a 4
× 250 mm RP-18 (10 μM) LiChrospher 100 column with a HPLC pump connected with a
variable wavelength monitor, a controller, and an integrator. ESI-TOF mass spectra of
nucleosides were recorded on a Micro-TOF spectrometer. Molecular masses of
oligonucleotides were determined by MALDI-TOF mass spectrometry in the linear positive
mode with 3-hydroxypicolinic acid (3-HPA) as a matrix (Table 1). Tm values were determined
from the melting curves using the software MELTWIN, version 3.0 (J. A. McDowell, 1996).
CD spectra were recorded with a Jasco 815 spectrophotometer. pKa values were determined
as described before.26
Acid-catalyzed hydrolysis of nucleosides monitored by HPLC
For the acid-catalyzed hydrolysis of nucleosides, a stock solution of the appropriate
nucleoside was prepared by dissolving 1 mg compound in 1 mL of 0.1 N HCl. At certain time
intervals, 100 µL of the stock solution was taken out and neutralized with 10 µL
triethylamine. This mixture was injected on a RP-18 HPLC column using the following
isocratic mixtures: (I) 0.1 M triethylammonium acetate/MeCN 99:1; (II) 0.1 M
triethylammonium acetate/MeCN 97:3. The hydrolysis was monitored at 260 nm with a UV
wavelength detector.
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C6OB00622A
Synthesis, purification, and characterization of oligonucleotides
The oligonucleotides were synthesized on an automated DNA synthesizer on a 1 μmol scale
employing standard phosphoramidites as well as 21, 22, and the phosphoramidites of 1b and
2a. The coupling yields were always higher than 95%. After cleavage from the solid support,
the oligonucleotides were deprotected in concentrated aqueous ammonia solution for 16 h at
55 °C. The purification of the “trityl-on” oligonucleotides was carried out on reversed-phase
HPLC using the following gradient system at 260 nm: (A) MeCN; (B) 0.1 M (Et3NH)OAc
(pH 7.0):MeCN, 95:5; gradient I: 0-3 min 10-15% A in B, 3-15 min 15−50% A in B, flow
rate 0.8 mL/min. The purified “trityl-on” oligonucleotides were treated with 2.5%
CHCl2COOH/CH2Cl2 for 2 min at 0 °C to remove the 4,4’-dimethoxytrityl residues. The
detritylated oligomers were purified again by reversed-phase HPLC with gradient II: 0-20
min 0-20% A in B, 20-25 min, 20% A in B, flow rate 0.8 mL/min. ODN-17 and ODN-18
were purified by reversed-phase HPLC using gradient III: 0-30 min 0-20% A in B. The
oligonucleotides were desalted on short column (RP-18) using water for elution of salt, while
the oligonucleotides were eluted with H2O/MeOH (2/3). The oligonucleotides were
lyophilized on a Speed-Vac evaporator to yield colorless solids which were frozen at -24°C.
Extinction coefficients ε260 (H2O) of nucleosides are dA, 15400; dG, 11700; dT, 8800; dC,
7300; MeiCd (1b), 61003c; iGd (2a), 43003g; iCF (1f), 51003a; MeiCOMe (3), 4600 (MeOH);
MeiCOProp (4), 4100 (MeOH).
General procedure for the sulfonylation of 1-(2-O-methyl-β-D-erythro-pentofuranosyl)-
5-methyluracil (5) with p-toluene sulfonyl chloride. Compound 59 (1.0 g, 3.7 mmol) was
dissolved in dry pyridine (20 mL). The solution was cooled to 0°C (ice-bath), then p-
toluenesulfonyl chloride (0.8 g, 4.2 mmol) was added and the solution was stirred overnight
at rt. A second portion of p-toluenesulfonyl chloride was introduced (0.7 g, 3.7 mmol) and
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Organic & Biomolecular Chemistry
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stirring continued for 3 h until the starting material was consumed (TLC-control, DOI: 10.1039/C6OB00622A
CH2Cl2/MeOH, 9:1). The solution was diluted with CH2Cl2 (50 mL) and the reaction was
quenched upon addition of water (20 mL). The aq. phase was extracted with CH2Cl2 (3 x 50
mL), the combined org. phase was washed with sat. NaHCO3 soln. (2 x 50 mL) and H2O (2 x
50 mL), dried (Na2SO4), the solvent was evaporated and the remaining residue was applied to
FC (silica gel, column 15 x 4 cm, CH2Cl2/MeOH, 95:5). Three products were isolated and the
analytical data are depicted below.
1-[2-O-Methyl-3,5-di-O-(p-toluenesulfonyl)-β-D-erythro-pentofuranosyl]-5-methyluracil
(6). From the fastest migrating zone compound 6 was isolated as colorless foam (443 mg,
21%). TLC (silica gel, CH2Cl2/MeOH, 95:5) Rf 0.70. λmax (MeOH)/nm 225 (ε/dm3 mol-1
cm-1 24900), 263 (9800). 1H NMR (DMSO-d6, 300 MHz) δ 1.76 (s, 3H, CH3), 2.41 (s, 3H,
CH3), 2.43 (s, 3H, CH3), 3.13 (s, 3H, OCH3), 3.98-4.15 (m, 4H, H-2’, H-4’, 2 x H-5’), 5.07 (t,
J = 5.1 Hz, 1H, H-3’), 5.66 (d, J = 5.7 Hz, 1H, H-1’), 7.43-7.50 (m, 5H, arom. H, H-6), 7.73-
7.90 (m, 4H, arom. H), 11.46 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ 12.0, 12.1, 21.1,
21.2, 57.8, 68.3, 75.5, 78.4, 78.5, 87.8, 110.2, 127.6, 127.8, 130.2, 130.3, 131.8, 132.2, 136.7,
145.3, 145.7, 150.4, 163.6. ESI-TOF m/z cald. for C25H28N2O10S2 [M+Na]+ 603.1078, found
603.1035.
1-[2-O-Methyl-3-O-(p-toluenesulfonyl)-β-D-erythro-pentofuranosyl]-5-methyluracil (7).
From the second zone compound 7 was isolated as colorless foam (218 mg, 14%). TLC
(silica gel, CH2Cl2/MeOH, 95:5): Rf 0.50. λmax (MeOH)/nm 263 (ε/dm3 mol-1 cm-1 10200). 1H
NMR (DMSO-d6, 300 MHz): δ 1.77 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H, OCH3),
3.34-3.39 (m, 1H, H-5’), 3.50-3.54 (m, 1H, H-5’), 3.95-3.96 (m, 1H, H-4’), 4.06 (dd, J = 5.4
Hz, J = 5.1 Hz, 1H, H-2’), 5.08- 5.10 (m, 1H, H-3’), 5.33 (m, 1H, HO-5’), 5.82 (d, J = 6.9
26

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Hz, 1H, H-1’), 7.51 (d, J = 8.4 Hz, 2H, arom. H), 7.66 (s, 1H, H-6), 7.87 (d, J = 8D.1OIH: 10z.1,0239H/C,6OB00622A
arom. H), 11.43 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ 12.0, 20.9, 57.4, 60.1, 77.0,
79.2, 82.5, 85.0, 109.9, 127.5, 130.0, 132.5, 135.4, 145.2, 150.4, 163.3. ESI-TOF m/z cald.
for C18H22N2O8S [M+Na]+ 449.0989, found 449.0987.
1-[2-O-Methyl-5-O-(p-toluenesulfonyl)-β-D-erythro-pentofuranosyl]-5-methyluracil (8).
From the slowest migrating zone compound 8 was isolated as colorless foam (540 mg, 34%).
TLC (silica gel, CH2Cl2/MeOH, 95:5) Rf 0.45. λmax (MeOH)/nm 266 (ε/dm3 mol-1 cm-1 9400).
1H NMR (DMSO-d6, 300 MHz) δ 1.78 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.33 (s, 3H, OCH3),
3.81 (t, J = 5.1 Hz, 1H, H-2’), 3.92-3.97 (m, 1H, H-4’), 4.01-4.07 (m, 1H, H-3’), 4.17-4.31
(m, 2H, H-5’), 5.39 (d, J = 6.3 Hz, 1H, HO-3’), 5.79 (d, J = 4.8 Hz, 1H, H-1’), 7.39 (s, 1H,
H-6), 7.48 (d, J = 8.4 Hz, 2H, arom. H), 7.84 (d, J = 12.6 Hz, 2H, arom. H), 11.40 (s, 1H,
NH). 13C NMR (DMSO-d6, 75 MHz) δ 11.8, 20.9, 57.4, 68.0, 69.5, 80.6, 81.1, 86.5, 109.7,
127.4, 129.9, 131.8, 135.5, 144.9, 150.1, 163.4. ESI-TOF m/z cald. for C18H22N2O8S
[M+Na]+ 449.0989, found 449.0977.
2,5’-Anhydro-2’-O-methyl-5-methyluridine (9). Method A (from compound 8).
Compound 8 (2.3 g, 5.4 mmol) was dissolved in dry CH3CN. Then, DBU (1,8-
diazabicyclo[5.4.0]undec-7-en, 2.2 mL, 2.2 g, 14.6 mmol) was added and the reaction
mixture was heated under reflux for 1 h. The solvent was evaporated and the remaining
residue was subjected to FC (silica gel, column 15 x 4 cm, CH2Cl2/MeOH, 9:1). Evaporation
of the main zone afforded compound 9 as colorless solid (722 mg, 53%). TLC (silica gel,
CH2Cl2/MeOH, 90:10) Rf 0.40. λmax (MeOH)/nm 248 (ε/dm3 mol-1 cm-1 9700). 1H NMR
(DMSO-d6, 300 MHz) δ 1.78 (s, 3H, CH3), 3.33 (s, 3H, OCH3), 4.04 (d, J = 12.9 Hz, 1H, H-
2’), 4.18 (d, J = 6.0 Hz, 1H, H-4’), 4.42 (t, J = 5.7 Hz, 2H, OCH2), 4.49 (m, 1H, H-3’), 5.09
27

Organic & Biomolecular Chemistry
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(d, J = 5.7 Hz, 1H, H-1’), 5.65 (s, 1H, HO-3’), 7.97 (s, 1H, H-6). 13C NMR (DMSO-d6, 7V5iew Article Online
DOI: 10.1039/C6OB00622A
MHz) δ 12.8, 57.9, 71.5, 73.9, 86.0, 87.5, 96.7, 117.2, 138.6, 156.5, 170.8. ESI-TOF m/z
cald. for C11H14N2O5 [M+Na]+ 277.0795, found 277.0797.
Method B (from compound 14). Compound 14 (570 mg, 1.92 mmol) was suspended in
MeOH (10 mL), then K2CO3 (0.020 g, 0.15 mmol) was added and the reaction mixture was
stirred for 20 min. The solvent was evaporated and the remaining residue was applied to FC
(silica gel, column 15 x 4 cm, CH2Cl2/MeOH, 95:5). Compound 9 was obtained as colorless
solid (396 mg, 81%). Analytical data were identical to the data described for method A.
1-[3-O-Acetyl-2-O-methyl-5-O-(p-toluenesulfonyl)-β-D-ribofuranosyl]uracil (13).
Compound 1211 (3.4 g, 10.8 mmol) was dissolved in dry pyridine (35 mL), p-toluenesulfonyl
chloride (7.0 g, 36.72 mmol) was added and the resulting mixture was stirred for 12 h at room
temperature. The reaction was quenched by the addition of ice-cold water (10 mL). Then,
CH2Cl2 (25 mL) was added and the organic layer was washed with saturated NaHCO3
solution (2 x 20 mL) and brine (2 x 20 mL). The organic phase was dried, filtrated and the
solvent was evaporated. The remaining residue was applied to FC (silica gel, column 15 x 4
cm, CH2Cl2/acetone, 4:1) furnishing compound 13 (3.9 g, 77%) as colorless foam. TLC
(silica gel, CH2Cl2/acetone, 4:1) Rf 0.60. λmax (MeOH)/nm 221 (ε/dm3 mol-1 cm-1 16100), 263
(11100). 1H NMR (DMSO-d6, 300 MHz) δ 1.78 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.41 (s, 3H,
CH3), 3.2 (s, 3H, OCH3), 4.11-4.36 (m, 4H, H-2’, H-4’, 2 x H-5’), 5.10 (m, 1H, H-3’), 5.78
(d, J = 6.1 Hz, 1H, H-1’), 7.46-7.48 (m, 3H, arom. H, H-6), 7.79-7.81 (m, 2H, arom. H),
11.45 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ 11.9, 20.3, 20.8, 57.9, 69.1, 69.6, 78.4,
79.0, 86.8, 110.0, 127.4, 129.9, 131.7, 135.7, 145.0, 150.2, 163.3, 169.3. ESI-TOF m/z cald.
for C20H24N2O9S [M+Na]+ 491.1095, found 491.1084.
28

Page 29 of 42
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C6OB00622A
3’-O-Acetyl-2,5’-anhydro-2’-O-5-dimethyluridine (14). Compound 13 (3.0 g, 6.4 mmol)
was dissolved in dry CH3CN (20 mL), then DBU (1,8-diazabicyclo[5.4.0]undec-7-en, 2.62
mL, 2.67 g, 17.5 mmol) was added and the reaction mixture was heated under reflux for 50
min. The solvent was evaporated and the remaining residue was applied to FC (silica gel,
column 15 x 4 cm, CH2Cl2/acetone, 1:1). Evaporation of the main zone afforded compound
14 as colorless solid (1.05 g, 55%). TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.60. λmax
(MeOH)/nm 248 (ε/dm3 mol-1 cm-1 10100). 1H NMR (DMSO-d6, 300 MHz) δ 1.81 (s, 3H,
CH3), 2.07 (s, 3H, CH3), 3.29 (s, 3H, OCH3), 4.12 (d, J = 12.9 Hz, 1H, H-5’), 4.41 (d, J = 6.0
Hz, 1H, H-4’), 4.52 (d, J = 12.9 Hz, 1H, H-5’), 4.68 (s, 1H, H-2’), 5.47 (d, J = 3.6 Hz, 1H, H-
1’), 5.77 (s, 1H, H-1’), 7.97 (s, 1H, H-6). 13C NMR (DMSO-d6, 75 MHz) δ 13.1, 20.5, 58.7,
73.5, 73.8, 84.4, 84.9, 96.5, 117.6, 138.7, 156.6, 169.7, 171.0. ESI-TOF m/z cald. for
C13H16N2O6 [M+Na]+ 319.0901, found 319.0911.
1-(2-O-Methyl-β-D-ribofuranosyl)-5-methyl-pyrimidin-4(3H)-one (3). Compound 9 (722
mg, 2.84 mmol) was dissolved in sat. NH3/MeOH (150 mL) and the solution was stirred for 6
h at 100 °C. The solvent was evaporated and the remaining residue was adsorbed on silica gel
and applied to FC (silica gel, column 15 x 4 cm, CH2Cl2/MeOH, 80:20). Evaporation of the
slower migrating zone afforded compound 3 as colorless solid (403 mg, 52%). TLC (silica
gel, CH2Cl2/MeOH, 80:20) Rf 0.30. λmax (MeOH)/nm 227 (sh) (ε/dm3 mol-1 cm-1 10200), 251
(sh) (4800), 260 (4600). 1H NMR (DMSO-d6, 300 MHz) δ 1.72 (s, 3H, CH3), 3.31 (s, 3H,
OCH3), 3.60 (m, 2H, H-5’), 3.91-3.95 (m, 2H, H-2’, H-4’), 4.20-4.21 (m, 1H, H-3’), 5.27 (d,
J = 4.8 Hz, 1H, HO-3’), 5.37 (t, J = 4.5 Hz, 1H, HO-5’), 5.52 (d, J = 6.9 Hz, 1H, H-1’), 6.87
(br s, 2H, NH2), 7.48 (s, 1H, H-6). 13C NMR (DMSO-d6, 75 MHz) δ 13.6, 57.5, 60.9, 68.3,
81.4, 86.3, 90.1, 114.4, 134.7, 154.5, 170.3. ESI-TOF m/z cald. for C11H17N3O5 [M+H]+
29